10-(3-cyclopropylaminomethyl-1-pyrrolidinyl)pyridobenzoxazinecarboxylic acid derivative effective against resistant bacterium

ABSTRACT

A compound as represented by the general formula (I) shown below exhibits high antibacterial activity against gram-positive bacteria, in particular, such drug-resistant bacteria as mRNA, PRSP and VRE:  
                 
wherein R1 is a methyl group, a fluoromethyl group, a methoxymethyl group, an acetoxymethyl group, a hydroxymethyl group or a methylene; R2 is a hydrogen atom, a lower alkyl group having 1 to 3 carbon atoms, or a pharmaceutically acceptable cation and an ester of a prodrug; R3 is a hydrogen atom or a halogen atom; R4 is a hydrogen atom, a lower alkyl group having 1 to 3 carbon atoms, a fluoromethyl group, a trifluoromethyl group or a fluorine atom; and R5 is a hydrogen atom or a fluorine atom, with exceptions where R1 is a methyl group, R4 and R5 are at the same time a hydrogen atom, and R3 is a fluorine atom.

TECHNICAL FIELD

The present invention relates to novel10-(3-cyclopropylaminomethyl-1-pyrrolidinyl)pyridobenzoxazine carboxylicacid derivatives, salts and hydrates thereof that, in addition to beingsafe and exhibiting strong antibacterial activities, are effectiveagainst drug-resistant bacteria that are less susceptible toconventional antibacterial agents.

TECHNICAL BACKGROUND

Reference should be made to the following articles: Japanese PatentLaid-Open Publication No. Sho 57-4698-6 (Patent Article 1); JapanesePatent Laid-Open Publication No. Sho 61-204188 (Patent Article 2);Japanese Patent Laid-Open Publication No. Sho 62-155282 (Patent Article3).

Since the development of norfloxacin, considerable effort has been madeworldwide to develop quinolone carboxylic acid-based antibacterialagents, which are also known as new quinolones and have now becomeimportant cures for infectious diseases.

The recent emergence of drug-resistant bacteria, such asMethicillin-Resistant Staphylococcus aureus (MRSA), Penicillin-ResistantStreptococcus pneumoniae (PRSP), and Vancomycin-Resistant Enterococcus(VRE), most of which are gram-positive bacteria, has posed a seriousthreat to the treatment of patients. Traditional quinolone carboxylicacid-based antibacterial agents have relatively weak antibacterialactivities against gram-positive bacteria and thus are not considered aseffective cures for the drug-resistant bacteria. Furthermore, theincreasing incidence of Quinolone-Resistant Staphylococcus aureus (QRSA)makes the use of these drugs even more difficult.

While pyridobenzoxazine carboxylic acid-based antibacterial agentssimilar to the ones claimed in the present invention are described in,for example, Patent Articles 1, 2, and 3, none of these agents offersufficient antibacterial activity against gram-positive bacteria, norare they described to have antibacterial activity against drug-resistantbacteria such as those described above.

DISCLOSURE OF THE INVENTION

It is therefore an objective of the present invention to provide novelpyridobenzoxazine carboxylic acid-based compounds that, in addition tobeing safe and exhibiting strong antibacterial activities, are effectiveagainst drug-resistant bacteria that are less susceptible toconventional antibacterial agents.

In view of the above-described problems, the present inventors havedevoted a significant amount of effort to seeking quinolone carboxylicacid derivatives that are effective against gram-positive bacteria, inparticular, such drug-resistant bacteria as MRSA, PRSP, and VRE, whichare less susceptible to traditional quinolone carboxylic acid-basedantibacterial agents. The effort was rewarded by the discovery of thecompounds of the present invention, which proved to be effective againstgram-positive bacteria, in particular, such drug-resistant bacteria asMRSA, PRSP, and VRE, and exhibit higher antibacterial activity ascompared not only with traditional quinolone carboxylic acid-basedantibacterial agents, but also with various other antibacterial agents.The discovery ultimately led the present inventors to complete thepresent invention.

According to the present invention, there is provided a compound asrepresented by the following general formula (I), or a salt or a hydratethereof:

wherein R1 is a methyl group, a fluoromethyl group, a methoxymethylgroup, an acetoxymethyl group, a hydroxymethyl group, or a methylenegroup; R2 is a hydrogen atom, a lower alkyl group having 1 to 3 carbonatoms, or a pharmaceutically acceptable cation and an ester of aprodrug; R3 is a hydrogen atom or a halogen atom; R4 is a hydrogen atom,a lower alkyl group having 1 to 3 carbon atoms, a fluoromethyl group, atrifluoromethyl group or a fluorine atom; and R5 is a hydrogen atom or afluorine atom.

Examples of the lower alkyl group in the general formula (I) include amethyl group, an ethyl group, a propyl group, an isopropyl group, and acyclopropyl group. Examples of the pharmaceutically acceptable cationinclude sodium ion, potassium ion, magnesium ion, calcium ion, andammonium ion. Examples of the ester of a prodrug include apivaloyloxymethyl group, an acetoxymethyl group, a phthalidinyl group,an indanyl group, a methoxymethyl group, and a5-methyl-2-oxo-1,3-dioxolene-4-yl group. Examples of the halogen atominclude fluorine, chlorine, bromine, and iodine.

BEST MODE FOR CARRYING OUT THE INVENTION

An exemplary production process of the compound of the present inventionwill now be described.

The compound of the present invention may be produced by reacting acompound represented by the following general formula (II):

[wherein R1 and R3 are the same as in the general formula (I); and R6 isrepresented by the following general formula (III):

[wherein R6 and R7 are each independently a fluorine atom, or a loweralkylcarbonyloxy group]]with a compound represented by the following general formula (IV), or anacid addition salt thereof:

[wherein R4 and R5 are the same as in the general formula (I); and R10is a hydrogen atom or a protective group of nitrogen atom such ast-butoxycarbonyl]and then removing the boron chelate and, if necessary, the protectivegroup of nitrogen atom.

The reaction of the compound of the general formula (II) with thecompound of the general formula (IV) may be carried out in the absenceor presence of a solvent, such as an alcohol, acetonitrile,dimethylsulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide,N-methylpyrrolidone, tetrahydrofuran, dioxane, benzene, or toluene, andin the presence of an acid receptor. The acid receptor may be acarbonate or a hydrogen carbonate of an alkali metal or an alkalineearth metal, or a basic organic compound, such as triethylamine,diazabicyclo-7-undecene, or pyridine. The reaction is typically carriedout at a temperature in the range of room temperature to 200° C. andpreferably in the range of 25° C. to 150° C. The reaction takes from 30min to 48 hours and is typically complete within 30 min to 15 hours.

If desired, the compound of the general formula (I) may be converted toits salt using an ordinary technique. Examples of such salts includesalts formed with an inorganic acid, such as hydrochloric acid, sulfuricacid, and phosphoric acid, salts formed with an organic acid, such asmethanesulfonic acid, lactic acid, oxalic acid, and acetic acid, andsalts formed with sodium, potassium, magnesium, calcium, aluminum,cerium, chromium, cobalt, copper, iron, zinc, platinum, silver, or thelike.

The compound of the present invention may be administered to humans oranimals in a pharmaceutically known form through a pharmaceuticallyknown route. For example, the compound may be prepared in the form ofpowders, tablets, capsules, ointments, injections, syrups, solutions,eye drops, and suppositories for oral or parenteral administration.

EXAMPLES

Exemplary tests as well as production processes for the compound of thepresent invention will now be described in detail with reference toexamples.

Reference Example 1Bis(acetato-O)[(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron

To a mixture of boric acid (12.8 g) and acetic anhydride (63.4 g), zincchloride (236 mg) was added and the resulting mixture was stirred atroom temperature for 0.5 hours. To this mixture,(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid ethyl ester (22.6 g) was added and the mixture was stirred at 60°C. for 2.5 hours. Subsequently, the reaction mixture was concentratedunder reduced pressure and the resulting residue was dissolved in ethylacetate (300 mL). The solution was sequentially washed with a saturatedaqueous solution of sodium hydrogen carbonate (2×200 mL) and then withwater (100 mL), followed by drying over anhydrous sodium sulfate andconcentration under reduced pressure. The resulting residue was purifiedon a silica gel column (dichloromethane: acetone=7:1), and the elutedyellow amorphous product was crystallized in an acetone/diethyl ethermixture to give 24.5 g ofbis(acetato-O)[(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron as a white powder.

¹H NMR(CDCl₃): δ 1.85 (s, 3H), 2.05 (s, 3H), 4.62 (ddd, J=2.9 Hz, 3.9Hz, 12.2 Hz, 1H), 4.74 (ddd, J=7.8 Hz, 10.3 Hz, 46.4 Hz, 1H), 4.90 (ddd,J=4.9 Hz, 10.3 Hz, 45.4 Hz, 1H), 4.92 (dd, J=1.0 Hz, 12.7 Hz, 1H),5.35-5.38 (m, 1H), 7.92 (dd, J=7.3 Hz, 9.3 Hz, 1H), 9.22 (s, 1H).

Elementary analysis (%): Calcd for C₁₇H₁₃BF₃NO₈.0.75H₂O: C, 46.34; H,3.32; N, 3.18; found: C, 46.30; H, 3.34; N, 3.30.

Reference Example 2 Synthesis ofbis(acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methoxymethyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron

Step 1:

(3S)-9,10-Difluoro-2,3-dihydro-3-hydroxymethyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid ethyl ester (1.30 g) was suspended in anhydrous dimethylformamide(40 mL). Silver oxide (I) (4.63 g) and methyl iodide (1.25 mL) were thenadded to the suspension. The resulting mixture was stirred at roomtemperature for 21 hours. Subsequently, insoluble materials were removedfrom the reaction mixture by filtration and the filtrate wasconcentrated under reduced pressure. The resulting residue was purifiedon a silica gel column (dichloromethane: acetone=5:1) to give 740 mg of(3S)-9,10-difluoro-2,3-dihydro-3-methoxymethyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid ethyl ester as a white powder.

MS(EI) m/z: 339 (M⁺).

Elementary analysis (%): Calcd for C₁₆H₁₅F₂NO₅: C, 56.64; H, 4.46; N,4.13; found: C, 56.56; H, 4.71; N, 4.26.

Step 2:

In a similar manner to Reference Example 1,(3S)-9,10-difluoro-2,3-dihydro-3-methoxymethyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid ethyl ester (679 mg) was reacted to give 830 mg of bis(acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methoxymethyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron as a colorless amorphous product.

¹H NMR(CDCl₃): δ 1.86 (s, 3H), 2.06 (s, 3H), 3.39 (s, 3H), 3.70 (dd,J=8.3 Hz, 10.3 Hz, 1H), 3.82 (dd, J=5.4 Hz, 10.3 Hz, 1H), 4.56 (dd,J=2.9 Hz, 12.2 Hz, 1H), 4.86 (dd, J=1.0 Hz, 12.2 Hz, 1H), 5.10-5.13 (m,1H), 7.89 (dd, J=7.3 Hz, 9.3 Hz, 1H), 9.13 (s, 1H).

Elementary analysis (%): Calcd for C₁₈H₁₆BF₂NO₉.1.5H₂O: C, 46.38; H,4.11; N, 3.00; found: C, 46.18; H, 3.74; N, 3.15.

Reference Example 3 Synthesis of bis(acetato-O)[(3S)-3-acetoxymethyl-9,10-difluoro-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron

Step 1:

(3S)-9,10-Difluoro-2,3-dihydro-3-hydroxymethyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid ethyl ester (976 mg) was suspended in anhydrous dichloromethane (30mL). To the suspension, acetic anhydride (368 mg) and4-dimethylaminopyridine (5.0 mg) were added and the resulting mixturewas refluxed for 1.5 hours while heated. Subsequently, the mixture wasallowed to cool and was washed with water. This was followed by dryingover anhydrous sodium sulfate and concentration under reduced pressure.The resulting residue was suspended in ethanol and the suspension wasfiltrated to give 1.04 g of(3R)-3-acetoxymethyl-9,10-difluoro-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid ethyl ester as a white powder.

MS(EI) m/z: 367(M⁺).

Elementary analysis (%): Calcd for C₁₇H₁₅F₂NO₆: C, 55.59; H, 4.12; N,3.81; found: C, 56.25; H, 4.15; N, 3.93.

Step 2:

In a similar manner to Reference Example 1,(3S)-3-acetoxymethyl-9,10-difluoro-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid ethyl ester (918 mg) was reacted to give 1.00 g of bis(acetato-O)[(3S)-3-acetoxymethyl-9,10-difluoro-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron as a colorless amorphous product.

¹H NMR(CDCl₃): δ 1.83 (s, 3H), 2.03 (s, 3H), 2.08 (s, 3H), 4.44-4.54 (m,2H), 4.63 (dd, J=2.9 Hz, 12.2 Hz, 1H), 4.89 (dd, J=1.0 Hz, 12.7 Hz, 1H),5.27-5.30 (m, 1H), 7.88 (dd, J=7.3 Hz, 9.3 Hz, 1H), 9.17 (s, 1H).

Elementary analysis (%): Calcd for C₁₉H₁₆BF₂NO₁₀.1.75H₂O: C, 45.76; H,3.94; N, 2.81; found: C, 45.94; H, 3.82; N, 2.95.

Reference Example 4 Synthesis oftrans-3-cyclopropylaminomethyl-4-methylpyrrolidine

Step 1:

trans-1-Benzyl-4-methyl-3-pyrrolidinecarboxylic acid (4.04 g) wasdissolved in dichloromethane (50 mL). To this solution,1,1′-carbonylbis-1H-imidazole (3.58 g) was added and the mixture wasstirred at room temperature for 1 hour. While the reaction mixture wascooled on an ice bath, a dichloromethane solution (15 mL) ofcyclopropylamine (1.53 mL) was added dropwise and the mixture wasstirred at room temperature for 3 hours. Subsequently, the reactionmixture was washed with water, was dried over anhydrous sodium sulfate,and was then concentrated under reduced pressure. The resulting residuewas crystallized in a hexane/diisopropyl ether mixture and the formedcrystal was filtrated. The collected crystal was then washed with ahexane/diisopropyl ether mixture and was dried under reduced pressure toobtain 4.07 g oftrans-1-benzyl-N-cyclopropyl-4-methyl-3-pyrrolidinecarboxamide as awhite crystal.

Melting point: 81-83° C.

MS (EI) m/z: 258(M⁺).

Step 2:

-   -   trans-1-Benzyl-N-cyclopropyl-4-methyl-3-pyrrolidinecarboxamide        (3.80 g) was suspended in anhydrous tetrahydrofuran (85 mL). To        this suspension, a lmol/L tetrahydrofuran solution of        borane-tetrahydrofuran complex (58.8 mL) was added and the        mixture was refluxed for 8 hours while heated. Subsequently, a 2        mol/L aqueous solution of sodium hydroxide (35 mL) was added to        the reaction mixture and the mixture was refluxed for 4 hours        while heated. After concentration under reduced pressure, the        resultant residue was extracted with toluene (2×100 mL) and the        toluene extracts were combined. The combined extract was washed        with water, was dried over anhydrous sodium sulfate, and was        then concentrated under reduced pressure. The resulting residue        was dissolved in dichloromethane (50 mL). To this solution,        di-tert-butyl dicarbonate (3.53 g) was added and the mixture was        stirred at room temperature for 4 hours. Subsequently, the        reaction mixture was concentrated under reduced pressure and the        resulting residue was purified on a silica gel column        (hexane:ethyl acetate=4:1 shifted to 1:1) to obtain 3.07 g of        trans-1-benzyl-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidine        as a colorless oil.

MS (FAB⁺) m/z: 345 (MH⁺).

HRMS (FAB⁺): Calcd for C₂₁H₃₃N₂O₂ (MH⁺): 345.2542; found: 345.2505.

Step 3:

-   -   trans-1-Benzyl-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidine        (3.00 g) was dissolved in ethanol (50 mL). To this solution,        7.5% palladium carbon (300 mg) was added and the mixture was        stirred at room temperature for 6 hours under a hydrogen        pressure of 3.9×10⁵ Pa. Subsequently, the catalyst was removed        from the reaction mixture by filtration and the collected        catalyst was washed with ethanol. The filtrate and the washing        solution were combined and the resulting residue was dried under        reduced pressure to obtain 2.12 g of        trans-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidine        as a pale brown oil.

MS (FAB⁺) m/z: 255 (MH⁺).

HRMS (FAB⁺): Calcd for C₁₄H₂₇N₂O₂ (MH⁺): 255.2073; found: 255.2079.

Step 4:

trans-3-[[(N-tert-Butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidine(2.07 g) was dissolved in dichloromethane (10 mL). While this solutionwas cooled on an ice bath, trifluoroacetic acid (5 mL) was added and themixture was stirred at room temperature for 2 hours. After concentrationunder reduced pressure, the resulting residue was dissolved intetrahydrofuran (6 mL) and the solution was allowed to stand for 13hours at room temperature. The separated crystal was collected byfiltration, followed by washing with tetrahydrofuran and drying underreduced pressure, to give 2.47 g oftrans-3-cyclopropylaminomethyl-4-methylpyrrolidine trifluoroacetic acidsalt. The salt product (2.37 g) was dissolved in water (5 mL), followedby addition of a 20% aqueous solution of sodium hydroxide to adjust thepH to 14. The solution was then extracted with diethyl ether (2×50 mL)and the extracts were combined. The combined extract was then dried overanhydrous sodium sulfate and was concentrated under reduced pressure.The resulting residue was purified by distillation under reducedpressure to obtain 660 mg oftrans-3-cyclopropylaminomethyl-4-methylpyrrolidine.

¹H NMR(CDCl₃): δ 0.30-0.37 (m, 2H), 0.41-0.45(m, 2H), 1.04(d, J=6.3 Hz,3H), 1.66-1.76(m, 4H), 2.08-2.13(m, 1H), 2.46(dd, J=7.3 Hz, 10.7 Hz,1H), 2.57(dd, J=8.3 Hz, 11.7 Hz, 1H), 2.63(dd, J=6.3 Hz, 10.7 Hz, 1H),2.80 (dd, J=5.4 Hz, 11.7 Hz, 1H), 3.10 (dd, J=6.8 Hz, 10.7 Hz, 1H), 3.14(dd, J=7.3 Hz, 10.7 Hz, 1H).

Elementary analysis (%): Calcd for C₉H₁₈N₂-2CF₃COOH: C 40.84, H 5.27, N7.33; found: C, 40.90; H, 5.47; N, 7.37.

Reference Example 5 Synthesis of(3R,4R)-3-cyclopropylaminomethyl-4-methylpyrrolidine

Step 1:

(3R,4R)-1-Benzyl-4-methyl-3-pyrrolidinecarboxylic acid (6.27 g) wassuspended in dichloromethane (250 mL). To this suspension,cyclopropylamine (1.76 mL) and hydrochloric acid1-ethyl-(3-dimethylaminopropyl)carbodiimide (12.2 g) were sequentiallyadded and the mixture was stirred at room temperature for 4 hours.Subsequently, the reaction mixture was washed with water, was dried overanhydrous sodium sulfate, and was then concentrated under reducedpressure. The resulting residue was purified on a silica gel column(ethyl acetate: methanol=10:1) to give 3.32 g of(3R,4R)-1-benzyl-N-cyclopropyl-4-methyl-3-pyrrolidinecarboxamide as awhite crystal.

MS (EI) m/z: 258 (M⁺).

Elementary analysis (%): Calcd for C₁₆H₂₂N₂O: C 74.38, H 8.58, N 10.84;found: C, 74.46; H, 8.67; N, 10.72.

Step 2:

In a similar manner to Step 2 in Reference Example 4,(3R,4R)-1-benzyl-N-cyclopropyl-4-methyl-3-pyrrolidinecarboxamide (5.52g) was reacted to give 4.16 g of(3R,4R)-1-benzyl-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidineas a pale brown oil.

MS (FAB⁺) m/z: 345 (MH⁺).

HRMS (FAB⁺): Calcd for C₂₁H₃₃N₂O₂ (MH⁺): 345.2542; found 345.2585.

Step 3:

In a similar manner to Step 3 in Reference Example 4,(3R,4R)-1-benzyl-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidine(4.00 g) was reacted to give 2.88 g of(3R,4R)-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidine.

MS (FAB⁺) m/z: 255 (MH⁺).

HRMS (FAB⁺): Calcd for C₁₄H₂₇N₂O₂(MH⁺): 255.2073; found: 255.2070.

Step 4:

In a similar manner to Step 4 in Reference Example 4,(3R,4R)-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidine(2.78 g) was reacted to give 730 mg of(3R,4R)-3-cyclopropylaminomethyl-4-methylpyrrolidine.

Specific rotation: +74.6° (c=0.648, methanol).

Elementary analysis (%): Calcd for C₉H₁₈N₂.2CF₃COOH: C 40.84, H 5.27, N7.33; found: C, 40.73; H, 5.26; N, 7.36.

Reference Example 6 Synthesis of(3S,4S)-3-cyclopropylaminomethyl-4-methylpyrrolidine

Step 1:

In a manner similar to Step 1 in Reference Example 5,(3S,4S)-1-benzyl-4-methyl-3-pyrrolidinecarboxylic acid (14.5 g) wasreacted to give 6.33 g of(3S,4S)-1-benzyl-N-cyclopropyl-4-methyl-3-pyrrolidinecarboxamide as apale brown crystal.

MS (EI) m/z: 258 (M⁺).

Elementary analysis (%): Calcd for C₁₆H₂₂N₂O: C 74.38, H 8.58, N 10.84;found: C, 74.64; H, 8.66; N, 10.71.

Step 2:

In a manner similar to Step 2 in Reference Example 4,(3S,4S)-1-benzyl-N-cyclopropyl-4-methyl-3-pyrrolidinecarboxamide (6.13g) was reacted to give 4.67 g of(3S,4S)-1-benzyl-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidineas a pale brown oil.

MS (FAB⁺) m/z: 345 (MH⁺).

HRMS (FAB⁺): Calcd for C₂₁H₃₃N₂O₂ (MH⁺): 345.2542; found: 345.2547.

Step 3:

In a similar manner to Step 3 in Reference Example 4,(3S,4S)-1-benzyl-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidine(4.47 g) was reacted to give 3.05 g of(3S,4S)-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidine.

MS (FAB⁺) m/z: 255 (MH⁺).

HRMS (FAB⁺): Calcd for C₁₄H₂₇N₂O₂ (MH⁺): 255.2073; found 255.2075.

Step 4:

In a similar manner to Step 4 in Reference Example 4,(3S,4S)-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-methylpyrrolidine(2.85 g) was reacted to give 1.21 g of(3S,4S)-3-cyclopropylaminomethyl-4-methylpyrrolidine.

Specific rotation: −74.5°(c=0.62, methanol).

Elementary analysis (%): Calcd for C₉H₁₁N₂-2CF₃COOH: C 40.84, H 5.27, N7.33; found: C, 40.80; H, 5.18; N, 7.39.

Reference Example 7 Synthesis ofcis-3-cyclopropylaminomethyl-4-methylpyrrolidine

Step 1:

-   -   c is-1-Benzyl-3-hydroxy-4-methylpyrrolidine (6.81 g) was        dissolved in dichloromethane (70 mL). While this solution was        cooled on a dry ice/acetone bath, triethylamine (5.21 mL) was        added. Methanesulfonyl chloride (2.89 mL) was then added        dropwise and the mixture was further stirred for 1 hour.        Following addition of water (50 mL), the temperature of the        mixture was allowed to rise to room temperature and the        dichloromethane layer was separated. The aqueous layer was        extracted with dichloromethane (50 mL) and the extract was        combined with the dichloromethane layer. The combined        dichloromethane layer was then washed with water, followed by        drying over anhydrous sodium sulfate and concentration under        reduced pressure. The resulting residue was dissolved in        acetonitrile (180 mL). To this solution, tetrabutylammonium        cyanide (23.9 g) was added and the mixture was refluxed for 7        hours while heated. Subsequently, the reaction mixture was        concentrated under reduced pressure and the resulting residue        was dissolved in ethyl acetate (300 mL). The solution was washed        with water, was dried over anhydrous sodium sulfate, and was        concentrated under reduced pressure. The resulting residue was        purified on a silica gel column (hexane:ethyl acetate=1:1) to        give 4.61 g of cis-1-benzyl-4-methyl-3-pyrrolidinecarbonitrile        as a brown oil.

IR (neat): 2240, 1496, 1454 cm⁻¹.

MS (EI) m/z: 200 (M⁺).

Step 2:

Lithium aluminum hydride (80%, 3.89 g) was suspended in diethyl ether(90 mL). While the suspension was cooled on an ice bath, a diethyl ethersolution (25 mL) of cis-1-benzyl-4-methyl-3-pyrrolidinecarbonitrile(4.11 g) was added dropwise and the mixture was stirred at roomtemperature for 1 hour. While the reaction mixture was cooled on an icebath, a saturated aqueous solution of sodium hydrogen carbonate (8 mL)was carefully added dropwise. Following dilution with diethyl ether (100mL), insoluble materials were collected by filtration and were washedwith diethyl ether. The filtrate and the washing solution were combinedand the combined solution was concentrated under reduced pressure. Theresulting residue was purified on a silica gel column (hexane:ethylacetate=1:1 shifted to ethyl acetate: methanol=10:1) to give 2.35 g ofcis-1-benzyl-4-methyl-3-aminomethylpyrrolidine as a pale yellow oil.

¹H NMR(CDCl₃): δ 0.94 (d, J=7.3 Hz, 3H), 1.09-1.66 (br, 2H), 2.03 (dd,J=7.3 Hz, 9.3 Hz, 1H), 2.11-2.26 (m, 2H), 2.31-2.42 (m, 1H), 2.58 (dd,J=8.3 Hz, 12.2 Hz, 1H), 2.82 (dd, J=5.9 Hz, 12.2 Hz, 1H), 2.96-3.02 (m,2H), 3.60 (s, 2H), 7.21-7.35 (m, 5H).

Step 3:

-   -   cis-1-Benzyl-4-methyl-3-aminomethylpyrrolidine (1000 mg) was        dissolved in methanol (10 mL). While this solution was cooled on        an ice bath, benzaldehyde (0.50 mL) was added dropwise and the        mixture was stirred at room temperature for 1 hour.        Subsequently, sodium cyanoborohydride (184 mg) was added and the        mixture was stirred at room temperature for 1.5 hours. This was        followed by a second addition of sodium cyanoborohydride (123        mg) and stirring for additional 5.5 hours. Subsequently, a 2        mol/L aqueous solution of sodium hydroxide (5 mL) was added to        the reaction mixture and the mixture was refluxed for 2 hours        while heated. Following concentration under reduced pressure,        the resulting residue was extracted with toluene (2×30 mL) and        the toluene extracts were combined. The combined toluene layer        was then washed with water, followed by drying over anhydrous        sodium sulfate and concentration under reduced pressure. The        resulting residue was purified on a silica gel column        (hexane:ethyl acetate=4:1) to give 690 mg of        cis-1-benzyl-3-benzylaminomethyl-4-methylpyrrolidine as a pale        yellow oil.

MS (EI) m/z: 294 (M⁺).

HRMS (EI): Calcd for C₂₀H₂₆N₂(M⁺): 294.2096; found: 294.2110.

Step 4:

-   -   cis-1-Benzyl-3-benzylaminomethyl-4-methylpyrrolidine (680 mg)        was dissolved in methanol (7 mL). To this solution, molecular        sieves 3A (700 mg), acetic acid (1.32 mL),        [1-(ethoxycyclopropyl)oxy]trimethylsilane (1.85 mL), and sodium        cyanoborohydride (435 mg) were added and the mixture was        refluxed for 4 hours while heated. Insoluble materials were        collected by filtration and were washed with methanol. The        filtrate and the washing solution were combined and the combined        organic layer was concentrated under reduced pressure. To the        resulting residue, water was added (5 mL), followed by addition        of a 2 mol/L aqueous solution of sodium hydroxide to make the        mixture basic. The mixture was then extracted with toluene (2×50        mL) and the extracts were combined. The combined toluene layer        was then washed with water, was dried over anhydrous sodium        sulfate, and was then concentrated under reduced pressure. The        resulting residue was purified on a silica gel column        (hexane:ethyl acetate=4:1) to give 648 mg of        cis-1-benzyl-3-(N-benzyl-N-cyclopropyl)aminomethyl-4-methylpyrrolidine        as a colorless oil.

MS (EI) m/z: 334 (M⁺).

HRMS (EI): Calcd for C₂₃H₃₀N₂(M⁺): 334.2409; found: 334.2403.

Step 5:

c is-1-Benzyl-3-(N-benzyl-N-cyclopropyl)aminomethyl-4-methylpyrrolidine(640 mg) was dissolved in ethanol (10 mL). To this solution, 10%palladium carbon (500 mg) and chloroform (0.77 mL) were added and themixture was stirred at 50° C. for 7 hours under a hydrogen pressure of3.9×10⁵ Pa. From the reaction mixture, the catalyst was collected byfiltration and was washed with ethanol. The filtrate and the washingsolution were combined and the combined organic layer was concentratedunder reduced pressure. To the resulting residue, water (2 mL) wasadded, followed by addition of a 2 mol/L aqueous solution of sodiumhydroxide to make the mixture basic. Sodium chloride was then added tothe mixture for salting out and the mixture was extracted with diethylether (2×25 mL). The diethyl ether extracts were combined and thecombined diethyl ether layer was dried over anhydrous sodium sulfate andwas concentrated under reduced pressure. The resulting residue waspurified on a silica gel column (hexane:ethyl acetate=4:1 shifted todichloromethane: methanol=10:1) to give 124 mg ofcis-3-cyclopropylaminomethyl-4-methylpyrrolidine as a pale brown oil.

MS (CI⁺) m/z: 155 (MH⁺).

HRMS (CI⁺): Calcd for C₉H₁₉N₂(MH⁺): 155.1548; found: 155.1553.

Reference Example 8 Synthesis of(3R,4S)-3-cyclopropylaminomethyl-4-methylpyrrolidine

Step 1:

(3R,4S)-1-Benzyl-3-hydroxy-4-methylpyrrolidine (4.00 g) was dissolved indichloromethane (40 mL). While this solution was cooled on a dryice/acetone bath, triethylamine (3.06 mL) was added. Methanesulfonylchloride (1.70 mL) was then added dropwise and the mixture was furtherstirred for 1 hour. Following addition of water (40 mL), the temperatureof the mixture was allowed to rise to room temperature and thedichloromethane layer was separated. The aqueous layer was extractedwith dichloromethane (40 mL) and the extract was combined with thedichloromethane layer. The combined dichloromethane layer was thenwashed with water, followed by drying over anhydrous sodium sulfate andconcentration under reduced pressure. The resulting residue wasdissolved in N,N-dimethylformamide (120 mL). To this solution,tetrabutylammonium cyanide (5.53 g) and sodium cyanide (2.05 g) wereadded and the mixture was stirred at 80° C. for 13 hours. Subsequently,the reaction mixture was concentrated under reduced pressure and water(50 mL) was added to the resulting residue. The mixture was extractedwith diethyl ether (2×200 mL). The diethyl ether extracts were combinedand the combined extract was washed with a saturated aqueous solution ofsodium chloride, followed by drying over anhydrous sodium sulfate andconcentration under reduced pressure. The resulting residue was purifiedon a silica gel column (hexane: ethyl acetate=4:1) to give 3.32 g of(3R,4S)-1-benzyl-4-methyl-3-pyrrolidinecarbonitrile as a brown oil.

¹H NMR(CDCl₃): δ 1.22 (d, J=7.3 Hz, 3H), 2.12 (dd, J=8.3 Hz, 9.3 Hz,1H), 2.45-2.57 (m, 1H), 2.60-2.67 (m, 1H), 2.99 (dd, J=7.3 Hz, 9.3 Hz,1H), 3.09-3.19 (m, 2H), 3.62 (s, 2H), 7.25-7.35 (m, 5H).

MS(EI) m/z: 200 (M⁺).

Step 2:

In a similar manner to Step 2 in Reference Example 7,(3R,4S)-1-benzyl-4-methyl-3-pyrrolidinecarbonitrile (3.20 g) was reactedto obtain 2.98 g of (3S,4S)-1-benzyl-4-methyl-3-aminomethylpyrrolidine.

¹H NMR(CDCl₃): δ 0.94 (d, J=7.3 Hz, 3H), 2.03 (dd, J=7.3 Hz, 9.3 Hz,1H), 2.11-2.26 (m, 2H), 2.31-2.43 (m, 1H), 2.58 (dd, J=8.3 Hz, 12.2 Hz,1H), 2.82 (dd, J=5.9 Hz, 12.2 Hz, 1H), 2.97-3.02 (m, 2H), 3.60 (s,2H),7.22-7.33 (m, 5H).

Step 3:

In a similar manner to Step 3 in Reference Example 7,(3S,4S)-1-benzyl-4-methyl-3-aminomethylpyrrolidine (2.80 g) was reactedto give 3.49 g of(3R,4S)-1-benzyl-3-benzylaminomethyl-4-methyl-pyrrolidine.

MS (EI) m/z: 294 (M⁺).

HRMS (EI): Calcd for C₂₀H₂₆N₂(M⁺): 294.2096; found: 294.2072.

Step 4:

In a similar manner to Step 4 in Reference Example 7,(3R,4S)-1-benzyl-3-benzylaminomethyl-4-methylpyrrolidine (3.40 g) wasreacted to give 3.72 g of(3R,4S)-1-benzyl-3-(N-benzyl-N-cyclopropyl)aminomethyl-4-methylpyrrolidine.

MS (FAB⁺) m/z: 335 (MH⁺).

HRMS (EI): Calcd for C₂₃H₃₁N₂(MH⁺): 335.2487; found: 335.2503.

Step 5:

In a similar manner to Step 5 in Reference Example 7,(3R,4S)-1-benzyl-3-(N-benzyl-N-cyclopropyl)aminomethyl-4-methylpyrrolidine(3.60 g) was reacted to give 1.29 g of(3R,4S)-3-cyclopropylaminomethyl-4-methylpyrrolidine.

MS (CI⁺) m/z: 155 (MH⁺).

HRMS (CI⁺): Calcd for C₉H₁₉N₂(MH⁺): 155.1548; found: 155.1539.

Reference Example 9 Synthesis of(3S,4R)-3-cyclopropylaminomethyl-4-methylpyrrolidine

Step 1:

In a similar manner to Step 1 in Example 8,(3S,4R)-1-benzyl-3-hydroxy-4-methylpyrrolidine (4.62 g) was reacted togive 3.07 g of (3S,4R)-1-benzyl-4-methyl-3-pyrrolidinecarbonitrile.

¹H NMR(CDCl₃): δ 1.22 (d, J=6.8 Hz, 3H), 2.13 (t,J=9.3 Hz, 1H),2.45-2.55 (m, 1H), 2.61-2.65 (m, 1H), 2.99 (dd, J=6.8 Hz, 9.3 Hz, 1H),3.09-3.19 (m, 2H), 3.62 (s, 2H), 7.27-7.34 (m 5H).

Step 2:

In a similar manner to Step 2 in Reference Example 7,(3S,4R)-1-benzyl-4-methyl-3-pyrrolidinecarbonitrile (3.00 g) was reactedto give 1.44 g of (3R,4R)-1-benzyl-4-methyl-3-aminomethylpyrrolidine.

MS (EI) m/z: 204 (M⁺).

HRMS (EI): Calcd for C₁₃H₂₀N₂(M⁺): 204.1626; found: 204.1614.

Step 3:

In a similar manner to Step 3 in Reference Example 7,(3R,4R)-1-benzyl-4-methyl-3-aminomethylpyrrolidine (1.06 g) was reactedto give 1.20 g of(3S,4R)-1-benzyl-3-benzylaminomethyl-4-methylpyrrolidine.

MS (EI) m/z: 294 (M⁺).

HRMS (EI): Calcd for C₂₀H₂₆N₂(M⁺): 294.2096; found: 294.2106.

Step 4:

In a similar manner to Step 4 in Reference Example 7,(3S,4R)-1-benzyl-3-benzylaminomethyl-4-methylpyrrolidine (1.40 g) wasreacted to give 1.55 g of(3S,4R)-1-benzyl-3-(N-benzyl-N-cyclopropyl)aminomethyl-4-methylpyrrolidine.

MS (FAB⁺) m/z: 335 (MH⁺).

HRMS (EI): Calcd for C₂₃H₃₁N₂(MH⁺): 335.2487; found: 335.2498.

Step 5:

In a similar manner to Step 5 in Reference Example 7,(3S,4R)-1-benzyl-3-(N-benzyl-N-cyclopropyl)aminomethyl-4-methylpyrrolidine(700 mg) was reacted to give 215 mg of(3S,4R)-3-cyclopropylaminomethyl-4-methylpyrrolidine.

MS (CI⁺) m/z: 155 (MH⁺).

HRMS (CI⁺): Calcd for C₉H₁₉N₂(MH⁺): 155.1548; found: 155.1510.

Reference Example 10 Synthesis oftrans-3-cyclopropylaminomethyl-4-trifluoromethylpyrrolidine

Step 1:

In a similar manner to Step 1 in Example 4,trans-1-benzyl-4-trifluoromethyl-3-pyrrolidinecarboxylic acid (3.00 g)was reacted to give 3.32 g oftrans-1-benzyl-4-trifluoromethyl-3-pyrrolidinecarboxamide.

¹H NMR(CDCl₃): δ 0.42-0.46 (m, 2H), 0.75-0.79 (m, 2H), 2.64-2.78 (m,4H), 2.82-2.86 (m, 1H), 2.95 (t, J=9.3 Hz, 1H), 3.10-3.22 (m, 1H), 3.59(d, J=13.2 Hz, 1H), 3.68 (d, J=12.7 Hz, 1H), 6.34-6.53 (br, 1H),7.26-7.36 (m, 5H).

Step 2:

In a similar manner Step 2 in Example 4,trans-1-benzyl-4-trifluoromethyl-3-pyrrolidinecarboxamide (3.21 g) wasreacted to give 3.37 g oftrans-1-benzyl-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-trifluoromethylpyrrolidine.

MS (FAB⁺) m/z: 399 (MH⁺).

HRMS (FAB⁺): Calcd for C₂₁H₃₀F₃N₂O₂ (MH⁺): 399.2259; found: 399.2254.

Step 3:

In a similar manner to Step 3 in Example 4,trans-1-benzyl-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-trifluoromethylpyrrolidine(3.27 g) was reacted to give 2.38 g oftrans-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-trifluoromethylpyrrolidine.

MS (FAB⁺) m/z: 309 (MH⁺).

HRMS (FAB⁺): Calcd for C₁₄H₂₄F₃N₂O₂(MH⁺): 309.1790; found: 309.1783.

Step 4:

In a similar manner to Step 4 in Example 4,trans-3-[[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl]-4-trifluoromethylpyrrolidine(2.30 g) was reacted to give 992 mg oftrans-3-cyclopropylaminomethyl-4-trifluoromethylpyrrolidine.

¹H NMR(CDCl₃): δ 0.29-0.33 (m, 2H), 0.42-0.46 (m, 2H), 2.10-2.15 (m,1H), 2.30-2.39 (m, 1H), 2.41-2.53 (m, 1H), 2.62-2.71 (m, 2H), 2.83 (dd,J=6.3 Hz, 11.7 Hz, 1H), 3.10 (d, J=6.8 Hz, 2H), 3.18 (dd, J=7.8 Hz, 11.7Hz, 1H).

Elementary analysis (%): Calcd for C₉H₁₅F₃N₂.2CF₃COOH: C 35.79, H 3.93,N 6.42; found: C, 35.82; H, 3.90; N, 6.59.

Reference Example 11 Synthesis of (3R,4S)-3-cyclopropylaminomethyl-4-fluoropyrrolidine (Process (I))

Step 1:

(E)-3-Benzyloxypropenyl-(1R)-camphorsultam (21.6 g) was dissolved indichloromethane (300 mL) containing trifluoroacetic acid (0.116 mL). Tothis solution, N-methoxymethyl-N-(trimethylsilyl)benzylamine (15.0 g)was added dropwise and the mixture was further stirred for 2 hours. Themixture was sequentially washed with a saturated aqueous solution ofsodium hydrogen carbonate (2×200 mL) and then with water (200 mL),followed by drying over anhydrous sodium sulfate and concentration underreduced pressure. The resulting pale yellow oil was dissolved in diethylether (150 mL) and the solution was allowed to stand for 18 hours atroom temperature. The crystal formed was collected by filtration, waswashed with diethyl ether, and was then dried under reduced pressure togive 11.5 g ofN-[[(3S,4R)-benzyl-4-benzyloxypyrrolidin-3-yl]carbonyl]-(2′S)-bornane-10,2-sultamas a white crystal. The filtrate and the washing solution were combinedand the combined organic layer was concentrated under reduced pressure.The resulting residue was purified on a silica gel column(eluant=cyclohexane:ethyl acetate=4:1) to obtain additional 8.48 g ofN-[[(3S,4R)-benzyl-4-benzyloxypyrrolidin-3-yl]carbonyl]-(2′S)-bornane-10,2-sultam.

¹H NMR(CDCl₃): δ 0.95 (s, 3H), 1.02 (s, 3H), 1.32-1.45 (m, 2H),1.86-1.96 (m, 3H), 2.00-2.10 (m, 2H), 2.57 (dd, J=9.3 Hz, 5.3 Hz), 2.69(dd, J=9.8 Hz, 3.9 Hz, 1H), 2.93 (dd, J=10.3 Hz, 6.3 Hz, 1H), 3.20 (t,J=9.3 Hz), 3.42-3.51 (m, 3H), 3.69-3.74 (m, 2H), 3.90 (d, J=11.7 Hz),4.54 (d, J=11.7 Hz), 4.63-4.66 (m, 1H), 7.22-7.31 (m, 10H).

Step 2:

Lithium aluminum hydride (80%, 5.56 g) was suspended in tetrahydrofuran(170 mL). While the suspension was cooled on a sodium chloride/ice bath,a tetrahydrofuran solution (300 mL) ofN-[[(3S,4R)-benzyl-4-benzyloxypyrrolidin-3-yl]carbonyl]-(2′S)-bornane-10,2-sultam(19.9 g) was added dropwise and the mixture was stirred at −5° C. orbelow for 1 hour. Subsequently, water (34 mL) was carefully addeddropwise to the mixture. Insoluble materials were collected byfiltration and were washed with ethyl acetate (2×400 mL). The filtrateand the washing solutions were combined and the combined organic layerwas extracted with 1 mol/L hydrochloric acid (2×500 mL). Thehydrochloric acid extracts were combined and a 30% aqueous solution ofsodium hydroxide was added to make the combined solution basic (pH 14).The mixture was then extracted with diethyl ether (2×500 mL) and thediethyl ether extracts were combined. The combined diethyl ether layerwas concentrated under reduced pressure and the resulting residue waspurified on a silica gel column (eluant=hexane:ethyl acetate=1:1) togive 9.91 g of (3R,4R)-(1-benzyl-4-benzyloxypyrrolidin-3-yl)methanol asa pale yellow oil.

¹H NMR(CDCl₃): δ 2.29-2.34(m, 1H), 2.40 (dd, J=10.3 Hz, 4.4 Hz, 1H),2.68 (dd, J=9.3 Hz, 2.4 Hz, 1H), 2.75 (dd, J=9.8 Hz, 6.3 Hz, 1H), 3.18(dd, J=9.8 Hz, 6.8 Hz, 1H), 3.61 (s, 2H), 3.65 (dd, J=10.3 Hz, 4.4 Hz,1H), 3.73 (dd, J=10.3 Hz, 4.4 Hz, 1H), 4.07 (ddd, J=6.3 Hz, 4.4 Hz, 2.0Hz, 1H), 4.48 (s, 2H), 7.25-7.35 (m, 10H).

Step 3:

Process (A): (3R,4R)-(1-benzyl-4-benzyloxypyrrolidin-3-yl)methanol (9.80g) was dissolved in ethanol (100 mL). To this solution, 10% palladiumcarbon (2.00 g) was added and the mixture was stirred at 50° C. for 21hours under a hydrogen pressure of 3.9×10⁵ Pa. Subsequently, thecatalyst was collected from the reaction mixture by filtration through aCelite pad. The collected catalyst and the Celite pad were washed withethanol. The filtrate and the washing solution were combined and thecombined organic layer was concentrated under reduced pressure. Theresulting residue was dissolved in ethanol (100 mL), followed byaddition of 10% palladium carbon (2.00 g). The mixture was then stirredat 50° C. for 20 hours under a hydrogen pressure of 3.9×10⁵ Pa.Subsequently, the catalyst was collected from the reaction mixture byfiltration through a Celite pad. The collected catalyst and the Celitepad were washed with ethanol. The filtrate and the washing solution werecombined and the combined organic layer was concentrated under reducedpressure. The resulting residue was dried under reduced pressure to give3.77 g of (3R,4R)-(4-hydroxypyrrolidin-3-yl)methanol.

¹H NMR (DMSO-d₆): δ 1.96-2.03 (m, 1H), 2.61 (dd, J=11.6 Hz, 5.5 Hz, 1H),2.68 (dd, J=11.6 Hz, 3.1 Hz, 1H), 2.91 (dd, J=11.1 Hz, 5.5 Hz, 1H), 3.06(dd, J=11.0 Hz, 7.3 Hz, 1H), 3.26 (dd, J=10.4 Hz, 7.3 Hz, 1H), 3.37 (dd,J=10.4 Hz, 6.1 Hz), 3.90-3.93 (m, 1H).

Sodium hydroxide (2.70 g) was dissolved in water (25 mL) and dioxane (15mL) was added. To this solution,(3R,4R)-(4-hydroxypyrrolidin-3-yl)methanol (1.00 g) was dissolved. Whilethe solution was cooled on an ice bath, carbobenzoxy chloride (0.97 mL)was added dropwise. The mixture was stirred at 5° C. or below for 1hour, followed by dropwise addition of carbobenzoxy chloride (0.97 mL).The mixture was further stirred at 5° C. or below for additional 1 hourand carbobenzoxy chloride (0.97 mL) was subsequently added dropwise.This was followed by stirring for 1 hour at 5° C. or below and another 1hour at room temperature. Subsequently, the reaction mixture wasextracted with dichloromethane (2×100 mL). The dichloromethane extractswere combined, and the combined dichloromethane layer was dried overanhydrous sodium sulfate and was concentrated under reduced pressure.The resulting residue was purified on a silica gel column(eluant=hexane:ethyl acetate=1:1 shifted to ethyl acetate:methanol=20:1) to give 1.18 g of(3R,4R)-[1-benzyloxycarbonyl-4-hydroxypyrrolidin-3-yl]methanol as amilky white tar-like product.

MS (EI) m/z: 251 (M⁺).

¹H NMR(CDCl₃): δ 2.08-2.40 (br +m, 2H), 2.58-2.79 (br, 1H), 3.20 (dd,J=11.0 Hz, 7.3 Hz, 1H), 3.32 (dt, J=11.1 Hz, 5.5 Hz, 1H), 3.59-3.76 (m,4H), 4.23-4.33 (br, 1H), 5.12 (s, 2H), 7.28-7.36 (m, 5H).

Process (B): (3R,4R)-[1-benzyl-4-benzyloxypyrrolidin-3-yl]methanol (10.0g) was dissolved in methanol (200 mL). To this solution, 10% palladiumcarbon (3.00 g) suspended in water (60 mL) and ammonium formate (21.2 g)were sequentially added, and the mixture was heat-refluxed for 4 hourswhile being stirred. Subsequently, the catalyst was collected from thereaction mixture by filtration through a Celite pad. The collectedcatalyst and the Celite pad were washed with a methanol/water mixture(80:20). The filtrate and the washing solution were combined and thecombined solution was concentrated under reduced pressure. The resultingpale brown, tar-like material was dissolved in N,N-dimethylformamide(100 mL). While this solution was cooled on an ice bath, triethylamine(9.40 mL) was added, followed by dropwise addition of carbobenzoxychloride (6.00 mL). While being cooled on an ice bath, the resultingmixture was stirred for 1.5 hours and was subsequently concentratedunder reduced pressure. The resulting residue was dissolved in ethylacetate (400 mL) and the solution was washed with a saturated aqueoussolution of sodium chloride (2×100 mL), was dried over anhydrous sodiumsulfate, and was then concentrated under reduced pressure. The resultingresidue was purified on a silica gel column (eluant=ethyl acetate,shifted to ethyl acetate: methanol=20:1) to give 7.66 g of(3R,4R)-[1-benzyloxycarbonyl-4-hydroxypyrrolidin-3-yl]methanol as amilky white tar-like product.

This compound was identical to the compound obtained by Process (A).

Step 4:

Process (A):(3R,4R)-(1-benzyloxycarbonyl-4-hydroxypyrrolidin-3-yl)methanol (3.19 g)was dissolved in N,N-dimethylformamide (91 mL). While this solution wascooled on an ice bath, imidazole (6.05 g) andtert-butylchlorodimethylsilane (5.74 g) were sequentially added and themixture was stirred at room temperature for 3 hours. Subsequently, thereaction mixture was concentrated under reduced pressure and theresulting residue was dissolved in diethyl ether (400 mL). The diethylether layer was washed with a saturated aqueous solution of sodiumchloride (2×100 mL), was dried over anhydrous sodium sulfate, and wasthen concentrated under reduced pressure. The resulting residue waspurified on a silica gel column (eluant=hexane:ethyl acetate=4:1) togive 5.46 g of(3R,4R)-1-benzyloxycarbonyl-3-(tert-butyldimethylsilyl)oxymethyl-4-(tert-butyldimethylsilyl)oxypyrrolidineas a colorless oil.

MS (CI⁺): m/z=480 (MH⁺).

¹H NMR(CDCl₃): δ 0.03 (s, 3H), 0.05 (s, 3H), 0.06 (s, 3H), 0.07 (s, 3H),0.87 (s, 9H), 0.88 (s, 9H), 2.17-2.27 (m, 1H), 3.21-3.28 (m, 2H),3.48-3.67 (m, 4H), 4.21-4.28 (m, 1H), 5.13 (s, 2H), 7.31-7.37 (m, 5H).

(3R,4R)-1-Benzyloxycarbonyl-3-(tert-butyldimethylsilyl)oxymethyl-4-(tert-butyldimethylsilyl)oxypyrrolidine(5.46 g) was dissolved in tetrahydrofuran (23 mL). While this solutionwas cooled on an ice bath, water (23 mL) and acetic acid (68 mL) weresequentially added and the mixture was stirred at room temperature for 8hours. Subsequently, the reaction mixture was concentrated under reducedpressure and the resulting residue was purified on a silica gel column(eluant=hexane:ethyl acetate=4:1 shifted to 1:1) to give 2.74 g of(3R,4R)-1-benzyloxycarbonyl-3-hydroxymethyl-4-(tert-butyldimethylsilyloxy)pyrrolidineas a colorless oil.

MS (CI⁺): m/z=366(MH⁺).

¹H NMR(CDCl₃): δ 0.07-0.08 (m, 6H), 0.88 (s, 9H), 2.23-2.35 (m, 1H),3.21-3.30 (m, 2H), 3.58-3.72 (m, 4H), 4.17-4.25 (m, 1H), 5.128 (s, 1H),5.135 (s, 1H), 7.31-7.37 (m, 5H).

(3R,4R)-1-Benzyloxycarbonyl-3-hydroxymethyl-4-(tert-butyldimethylsilyloxy)pyrrolidine(2.73 g) was dissolved in dichloromethane (60 mL). While this solutionwas cooled on a sodium chloride/ice bath, triethylamine (1.21 mL) wasadded, which was followed by dropwise addition of methanesulfonylchloride (0.71 mL) at −5° C. or below. The reaction mixture was thenstirred at −5° C. or below for 1 hour, was washed with water (2×25 mL),was dried over anhydrous sodium sulfate, and was then concentrated underreduced pressure. The resulting residue was dissolved inN,N-dimethylformamide (60 mL), followed by addition of sodium azide(1.14 g) and stirring at 100° C. for 2 hours. The reaction mixture wasthen concentrated under reduced pressure and water (30 mL) was added tothe resulting residue. The mixture was then extracted with diethyl ether(2×100 mL) and the diethyl ether extracts were combined. The combineddiethyl ether layer was dried over anhydrous sodium sulfate and wasconcentrated under reduced pressure. The resulting residue was purifiedon a silica gel column (eluant=hexane:ethyl acetate=4:1) to give 3.06 gof(3R,4R)-3-azidomethyl-1-benzyloxycarbonyl-4-(tert-butyldimethylsilyl)oxypyrrolidineas a colorless oil.

MS (CI⁺):m/z=391 (MH⁺).

¹H NMR(CDCl₃): δ 0.07-0.09 (m, 3H), 2.23-2.34 (m, 1H), 3.19-3.25 (m,2H), 3.27-3.40 (m, 2H), 3.60-3.71 (m, 2H), 4.11-4.17 (m, 1H), 5.13 (s,2H), 7.31-7.37 (m, 5H).

(3R,4R)-3-Azidomethyl-1-benzyloxycarbonyl-4-(tert-butyldimethylsilyl)oxypyrrolidine(3.05 g) was dissolved in tetrahydrofuran (50 mL). While this solutionwas cooled on an ice bath, tetrabutylammonium fluoride (lmol/Ltetrahydrofuran solution, 13.3 mL) was added dropwise and the mixturewas stirred for additional 1 hour. Subsequently, a saturated aqueoussolution of sodium chloride 70 mL) was added and the mixture wasextracted with ethyl acetate (150 mL, 100 mL). The ethyl acetateextracts were combined and the combined solvent was dried over anhydroussodium sulfate and was concentrated under reduced pressure. Theresulting residue was purified on a silica gel column (eluant=ethylacetate) to give 2.01 g of(3R,4R)-3-azidomethyl-1-benzyloxycarbonyl-4-hydroxypyrrolidine as amilky white syrup-like product.

MS (CI⁺):m/z=277 (MH⁺).

¹H NMR (CDCl₃): δ 2.18-2.30 (br, AH), 2.32-2.40 (m, 1H), 3.24 (dd,J=11.6 Hz, 6.1 Hz, 1H), 3.30-3.47 (m, 3H), 3.68-3.75 (m, 2H), 4.18-4.24(m, 1H), 5.13 (s, 2H), 7.31-7.37 (m, 5H).

Process (B):(3R,4R)-[1-Benzyloxycarbonyl-4-hydroxypyrrolidin-3-yl]methanol (3.00 g),sodium azide (2.32 g), triphenylphosphine (3.43 g) andN,N-dimethylformamide (60 mL) were mixed with each other. While themixture was cooled on an ice bath, a dichloromethane solution (14 mL) ofcarbon tetrabromide (4.34 g) was added dropwise. The reaction mixturewas stirred for 25 hours at room temperature and additional 2 hours at60° C., followed by addition of methanol (5 mL) and concentration underreduced pressure. The resulting residue was dissolved in ethyl acetate(200 mL) and was washed with a saturated aqueous solution of sodiumchloride (2×50 mL), followed by drying over anhydrous sodium sulfate andconcentration under reduce pressure. The resulting residue was purifiedon silica gel column (eluant=ethyl acetate:hexane=2:1) to give 2.94 g of(3R,4R)-3-azidomethyl-1-benzyloxycarbonyl-4-hydroxypyrrolidine as a palebrown syrup-like product. This compound was identical to the compoundobtained by Process (A).

Process (C):(3R,4R)-[1-Benzyloxycarbonyl-4-hydroxypyrrolidin-3-yl]methanol (150 mg)was dissolved in dichloromethane (12 mL) and 2,4,6-collidine (0.79 mL)was added. While this solution was cooled on an ice bath,methanesulfonyl chloride (46.2 μL) was added dropwise. The mixture wasthen stirred for 2 hours on the ice bath and was allowed to stand for 15hours in a refrigerator (3° C.). Subsequently, the reaction mixture wassequentially washed with water (2 mL), 1 mol/L hydrochloric acid (2×2mL), and a saturated aqueous solution of sodium chloride (2×2 mL),followed by drying over anhydrous sodium sulfate and concentration underreduced pressure. The resulting residue was purified on a silica gelcolumn (eluant=hexane:ethyl acetate=1:2 shifted to ethyl acetate) togive 38.7 mg of(3R,4R)-1-benzyloxycarbonyl-3-methanesulfonyloxy-4-methanesulfonyloxymethylpyrrolidineas a pale yellow syrup-like product and 133 mg of(3R,4R)-1-benzyloxycarbonyl-3-hydroxy-4-methanesulfonyloxymethylpyrrolidineas a white syrup-like product.

(3R,4R)-1-Benzyloxycarbonyl-3-hydroxy-4-methanesulfonyloxymethylpyrrolidine(125 mg) was dissolved in N,N-dimethylformamide (3 mL) and sodium azide(50.0 mg) was added. The mixture was stirred at 100° C. for 1 hour andwas then concentrated under reduced pressure. The resulting residue wasdissolved in ethyl acetate (5 mL) and the solution was washed with water(2×1 mL), followed by drying over anhydrous sodium sulfate andconcentration under reduced pressure. The resulting residue was purifiedon a silica gel column (eluant=ethyl acetate) to give 91.0 mg of(3R,4R)-3-azidomethyl-1-benzyloxycarbonyl-4-hydroxypyrrolidine as amilky white syrup-like product. The compound was identical to thecompound obtained by Process (A).

Step 5:

Process (A):(3R,4R)-3-Azidomethyl-1-benzyloxycarbonyl-4-hydroxypyrrolidine (1.20 g)was dissolved in dichloromethane (40 mL). While this solution was cooledon a sodium chloride/ice bath, diethylaminosulfur trifluoride (1.20 mL)was added dropwise and the mixture was stirred at room temperature for 3hours. The reaction vessel was again cooled on a sodium chloride/icebath and diethylaminosulfur trifluoride (0.57 mL) was again addeddropwise. The mixture was then stirred at room temperature for 2 hours.While the reaction mixture was cooled on an ice bath, a saturatedaqueous solution of sodium hydrogen carbonate (40 mL) was added dropwiseand the dichloromethane layer was separated. The dichloromethane layerwas sequentially washed with a saturated aqueous solution of sodiumhydrogen carbonate (2×20 mL) and water (20 mL), followed by drying overanhydrous sodium sulfate and concentration under reduced pressure. Theresulting residue was purified on a silica gel column(eluant=hexane:ethyl acetate=2:1) to give 726 mg of(3R,4S)-3-azidomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine as a palebrown oil.

MS (CI⁺):m/z=279 (MH⁺).

¹H NMR(CDCl₃): δ 2.34-2.54 (m, 1H), 3.22 (dt, J=11.0 Hz, 2.4 Hz, 1H),3.39-3.49 (m, 1H), 3.54-3.69 (m, 2H), 3.73-3.91 (m, 2H), 5.14 (s, 2H),5.16 (dt, J=53.2 Hz, 3.7 Hz, 1H), 7.32-7.37 (m, 5H).

Process (B):(3R,4R)-3-Azidomethyl-1-benzyloxycarbonyl-4-hydroxypyrrolidine (1.79 g)was dissolved in toluene (56 mL). While this solution was cooled on anice bath, 1,8-diazabicyclo[5.4.0]undec-7-ene (2.03 mL) was added. Thiswas followed by dropwise addition of perfluoro-1-octanesulfonyl fluoride(2.80 mL) and stirring for another 1 hour. Insoluble materials wereremoved from the reaction mixture by filtration and were washed withtoluene. The filtrate and the washing solution were combined and thecombined organic layer was concentrated under reduced pressure. Theresulting residue was then purified on a silica gel column(eluant=hexane:ethyl acetate=2:1) to give 1.58 g of(3R,4S)-3-azidomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine as a palebrown syrup-like product. The compound was identical to the compoundobtained by Process (A).

Step 6:

(3R,4S)-3-Azidomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine (1.35 g)was dissolved in ethanol (30 mL). To this solution, platinum oxide (IV)(190 mg) was added and the mixture was stirred at room temperature for 2hours in a stream of hydrogen (provided from a balloon). Subsequently,the catalyst was collected from the reaction mixture by filtrationthrough a Celite pad. The collected catalyst and the Celite pad werewashed with ethanol. The filtrate and the washing solution were combinedand the combined organic layer was concentrated under reduced pressure.The resulting residue was purified on a silica gel column (eluant=ethylacetate: methanol=10:1) to give 1.13 g of(3S,4S)-3-aminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine as a palebrown oil.

MS (CI⁺):m/z=253 (MH⁺).

Step 7:

(3S,4S)-3-Aminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine (1.10 g)was dissolved in methanol (13 mL). To this solution, molecular sieves 4A(440 mg) and benzaldehyde (0.44 mL) were sequentially added and themixture was stirred at room temperature for 1 hour. Subsequently, aborane-pyridine complex (0.44 mL) was added and the mixture was furtherstirred at room temperature for 3.5 hours. This was followed by additionof 6 mol/L hydrochloric acid (7.3 mL) and stirring at room temperaturefor 1 hour. Subsequently, a 30% aqueous solution of sodium hydroxide wasadded to make the mixture basic and the mixture was extracted withdiethyl ether (2×100 mL). The diethyl ether extracts were combined andthe combined diethyl ether layer was dried over anhydrous sodium sulfateand was then concentrated under reduced pressure. The resulting residuewas purified on a silica gel column (eluant=hexane:ethyl acetate=4:1shifted to 1:1) to give 1.18 g of(3S,4S)-3-benzylaminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine as acolorless tar-like product.

MS (CI⁺):m/z=343 (MH⁺).

Step 8:

(3S,4S)-3-Benzylaminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine(1.15 g) was dissolved in methanol (21 mL). To this solution, molecularsieves 3A (1.05 g), acetic acid (1.92 mL),[(1-ethoxycyclopropyl)oxy]trimethylsilane (2.70 mL), and sodiumcyanoborohydride (633 mg) were added and the mixture was heat-refluxedfor 2 hours while being stirred. Subsequently, insoluble materials wereremoved from the reaction mixture by filtration through a Celite pad.The insoluble materials and the Celite pad were washed with methanol.The filtrate and the washing solution were combined and a 2 mol/Laqueous solution of sodium hydroxide was added to make the combinedorganic layer basic (pH14). Methanol was then removed under reducedpressure and the residue was extracted with diethyl ether (2×100 mL).The diethyl ether extracts were combined and the combined diethyl etherlayer was dried over anhydrous sodium sulfate and was then concentratedunder reduced pressure. The resulting residue was purified on a silicagel column (eluant=hexane:ethyl acetate=4:1) to give 1.26 g of(3S,4S)-3-(N-benzyl-N-cyclopropyl)aminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidineas a colorless tar-like product.

MS (EI) m/z:=382 (M⁺).

Step 9:

(3S,4S)-3-(N-Benzyl-N-cyclopropyl)aminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine(1.22 g) was dissolved in ethanol (14 mL). To this solution, 10%palladium carbon (150 mg) was added and the mixture was stirred at roomtemperature for 4 hours in a stream of hydrogen (provided from aballoon). Subsequently, the catalyst was collected from the reactionmixture by filtration through a Celite pad. The collected catalyst andthe Celite pad were washed with ethanol. The filtrate and the washingsolution were combined and the combined organic layer was concentratedunder reduced pressure. The resulting residue was purified on a silicagel column (eluant=ethyl acetate: methanol=20:1). The eluate wasdistilled under reduced pressure to give 414 mg of(3R,4S)-3-cyclopropylaminomethyl-4-fluoropyrrolidine as a colorless oil.

MS (CI⁺): m/z=159 (MH⁺).

HRMS (CI⁺): Calcd for C₈H₁₆FN₂: 159.1298; found: 159.1316.

Reference Example 12 Synthesis of(3R,4S)-3-cyclopropylaminomethyl-4-fluoropyrrolidine (Process (II))

Step 1:

(3R,4R)-(4-Hydroxypyrrolidin-3-yl)methanol (1.18 g) was dissolved inethanol (25 mL) and triethylamine (1.40 mL) was added to the solution.While this mixture was cooled on a sodium chloride/ice bath, benzylbromide (1.10 mL) was added dropwise. The mixture was then stirred atroom temperature for 1 hour and was concentrated under reduced pressure.The resulting residue was purified on a silica gel column (eluant=ethylacetate: methanol=20:1) to give 1.02 g of(3R,4R)-(1-benzyl-4-hydroxypyrrolidin-3-yl)methanol as a milky whitesyrup-like product.

MS (EI⁺): m/z=207 (M⁺).

HRMS (EI⁺): Calcd for C₁₂H₁₇NO₂: 207.1259; found: 207.1237.

Step 2:

(3R,4R)-(1-Benzyl-4-hydroxypyrrolidin-3-yl)methanol (1.36 g) wasdissolved in dichloromethane (14 mL). While this solution was cooled onan dry ice/acetone bath, triethylamine (0.83 mL) was added, followed bydropwise addition of methanesulfonyl chloride (0.46 mL) and stirring for30 min. Water (10 mL) was then added to the reaction mixture and thetemperature of the mixture was allowed to rise to room temperature. Themixture was then diluted with dichloromethane (20 mL) and thedichloromethane layer was collected. The collected dichloromethane layerwas washed with water (2×10 mL), was dried over anhydrous sodiumsulfate, and was then concentrated under reduced pressure. The resultingresidue was purified on a silica gel column (hexane:ethyl acetate=1:1shifted to ethyl acetate: methanol=20:1). From a fraction eluted athexane:ethyl acetate=1:1, 585 mg of(3R,4R)-1-benzyl-3-methanesulfonyloxy-4-methanesulfonyloxymethylpyrrolidinewas obtained as a milky white syrup-like product.

MS (EI⁺): m/z=363 (M⁺).

HRMS (EI⁺): Calcd for C₁₄H₂₁NO₆S₂: 363.0810; found: 363.0804.

Also, 840 mg of(3R,4R)-1-benzyl-3-hydroxy-4-methanesulfonyloxymethylpyrrolidine wasobtained as a white crystal from a fraction eluted at ethyl acetate:methanol=20:1.

MS (EI⁺): m/z=285 (M⁺).

HRMS (EI⁺): Calcd for C₁₃H₁₉NO₄S: 285.1035; found: 285.1045.

Step 3:

(3R,4R)-1-Benzyl-3-hydroxy-4-methanesulfonyloxymethylpyrrolidine (835mg), sodium azide (381 mg), and N,N-dimethylformamide (12 mL) were mixedwith one another and the mixture was stirred at 120° C. for 1 hour.Subsequently, the reaction mixture was concentrated under reducedpressure. To the resulting residue, water (10 mL) was added and themixture was extracted with diethyl ether (2×30 mL). The diethyl etherextracts were combined and the combined extract was dried over anhydroussodium sulfate and was concentrated under reduced pressure. Theresulting residue was purified on a silica gel column (eluant=ethylacetate: methanol=20:1) to give 576 mg of(3R,4R)-3-azidomethyl-1-benzyl-4-hydroxypyrrolidine as a pale brown oil.

MS (EI⁺): m/z=232 (M⁺).

HRMS (EI⁺): Calcd for C₁₂H₁₆N₄₀: 232.1324; found: 232.1309.

Step 4:

(3R,4R)-3-Azidomethyl-1-benzyl-4-hydroxypyrrolidine (566 mg) wasdissolved in dichloromethane (9 mL). While this solution was cooled onan ice bath, diethylaminosulfur trifluoride (0.39 mL) was added dropwiseand the mixture was stirred at room temperature for 2 hours. While thereaction vessel was cooled on an ice bath, a saturated aqueous solutionof sodium hydrogen carbonate (9 mL) was added, and the mixture wasdiluted with dichloromethane (15 mL). The dichloromethane layer wascollected and the collected dichloromethane layer was washed with asaturated aqueous solution of sodium hydrogen carbonate (10 mL) and thenwater (10 mL), was dried over anhydrous sodium sulfate, and wasconcentrated under reduced pressure. The resulting residue was purifiedon a silica gel column (eluant=hexane:ethyl acetate=4:1). From the firsthalf fraction, 76.7 mg of(3R,4R)-3-azidomethyl-1-benzyl-4-fluoropyrrolidine was obtained as apale brown oil.

MS (EI⁺):m/z=234 (M⁺).

HRMS (EI⁺): Calcd for C₁₂H₁₅FN₄: 234.1281; found: 234.1263.

From the second half fraction, 220 mg of(3R,4S)-3-azidomethyl-1-benzyl-4-fluoropyrrolidine was obtained as apale brown oil.

MS (EI⁺): m/z=234 (M⁺).

HRMS (EI⁺): Calcd for C₁₂H₁₅FN₄: 234.1281; found: 234.1269.

Step 5:

(3R,4S)-3-Azidomethyl-1-benzyl-4-fluoropyrrolidine (215 mg) wasdissolved in ethanol (3 mL). To this solution, platinum oxide (IV) (30.0mg) was added and the mixture was stirred at room temperature for 5hours in a stream of hydrogen (provided from a balloon). Subsequently,the catalyst was removed from the reaction mixture by filtration througha Celite pad. The removed catalyst and the Celite pad were washed withethanol. The filtrate and the washing solution were combined and thecombined organic layer was concentrated under reduced pressure to obtain191 mg of (3S,4S)-3-aminomethyl-1-benzyl-4-fluoropyrrolidine as a brownoil.

MS (CI⁺): m/z=209 (MH⁺).

HRMS (CI⁺): Calcd for C₁₂H₁₈FN₂: 209.1454; found: 209.1465.

Step 6:

(3S,4S)-3-Aminomethyl-1-benzyl-4-fluoropyrrolidine (186 mg) wasdissolved in methanol (4 mL). To this solution, molecular sieves 4A(80.0 mg) and benzaldehyde (90.8 μL) were sequentially added and themixture was stirred at room temperature for 1 hour. Subsequently, aborane-pyridine complex (90.2 μL) was added and the mixture was furtherstirred at room temperature for 3 hours. This was followed by additionof 6 mol/L hydrochloric acid (1.5 mL) and stirring for 1 hour.Subsequently, a 6 mol/L aqueous solution of sodium hydroxide was addedto make the mixture basic and the mixture was extracted with diethylether (3×10 mL). The diethyl ether extracts were combined and thecombined diethyl ether layer was dried over anhydrous sodium sulfate andwas then concentrated under reduced pressure. The resulting residue waspurified on a silica gel column (eluant=hexane:ethyl acetate=4:1) togive 179 mg of (3S,4S)-1-benzyl-3-benzylaminomethyl-4-fluoropyrrolidineas a pale brown oil.

MS (CI⁺): m/z=299 (MH⁺).

HRMS (CI⁺): Calcd for C₁₉H₂₄FN₂: 299.1924; found: 299.1960.

Step 7:

(3S,4S)-1-Benzyl-3-benzylaminomethyl-4-fluoropyrrolidine (175 mg) wasdissolved in methanol (2 mL). To this solution, molecular sieves 3A(180mg), acetic acid (0.36 mL), [(1-ethoxycyclopropyl)oxy]trimethylsilane(0.47 mL) and sodium cyanoborohydride (110 mg) were added and themixture was heat-refluxed for 3 hours while being stirred. Subsequently,insoluble materials were removed from the reaction mixture by filtrationthrough a Celite pad. The insoluble materials and the Celite pad werewashed with methanol. The filtrate and the washing solution werecombined and a 2 mol/L aqueous solution of sodium hydroxide was added tomake the combined organic layer basic (pH14). Methanol was then removedunder reduced pressure and the residue was extracted with diethyl ether(3×100 mL). The diethyl ether extracts were combined and the combineddiethyl ether layer was dried over anhydrous sodium sulfate and was thenconcentrated under reduced pressure. The resulting residue was purifiedon a silica gel column (eluant=hexane:ethyl acetate=4:1) to give 172 mgof(3R,4S)-3-(N-benzyl-N-cyclopropyl)aminomethyl-1-benzyl-4-fluoropyrrolidineas a colorless tar-like product.

MS (CI⁺):m/z=339 (MH⁺).

HRMS (CI⁺): Calcd for C₂₂H₂₈FN₂: 339.2237; found: 339.2285.

Step 8:

(3R,4S)-3-(N-Benzyl-N-cyclopropyl)aminomethyl-1-benzyl-4-fluoropyrrolidine(170 mg) was dissolved in ethanol (10 mL). To this solution, 10%palladium carbon (200 mg) and chloroform (0.17 mL) were added and themixture was stirred at 50° C. for 23 hours under a hydrogen pressure of3.9×10⁵ Pa. Subsequently, palladium carbon was removed from the reactionmixture by filtration through a Celite pad. The removed palladium carbonand the Celite pad were washed with ethanol. The filtrate and thewashing solution were then combined and the combined organic layer wasconcentrated under reduced pressure. To the resulting residue, a 30%aqueous solution of sodium hydroxide (approximately 1 mL) was added.Subsequently, sodium chloride was added to saturation and the mixturewas extracted with diethyl ether (3×10 mL). The diethyl ether extractswere combined and the combined diethyl ether layer was dried overanhydrous sodium sulfate and was then concentrated under reducedpressure to give 65.4 mg of(3R,4S)-3-cyclopropylaminomethyl-4-fluoropyrrolidine as a pale brownoil. This compound was identical to the compound obtained in ReferenceExample 11 (Process (I)).

Reference Example 13 Synthesis of(3R,4R)-3-cyclopropylaminomethyl-4-fluoropyrrolidine

Step 1:

(3R,4R)-[1-Benzyloxycarbonyl-4-hydroxypyrrolidin-3-yl]methanol (2.50 g),triphenylphosphine (5.74 g), and benzoic acid (2.55 g) were dissolved intetrahydrofuran (60 mL). While this solution was cooled on a sodiumchloride/ice bath, azodicarboxylic acid diethyl ester (40% toluenesolution, 9.53 mL) was added dropwise. The mixture was stirred for 1hour at 0° C. or below and then for additional 2 hours at roomtemperature and was subsequently concentrated under reduced pressure.The resulting residue was purified on a silica gel column(eluant=hexane:ethyl acetate=2:1). The eluted pale brown tar-likematerial was dissolved in ethanol (60 mL). To this solution, potassiumcarbonate (4.07 g) dissolved in water (30 mL) was added and the mixturewas heat-refluxed for 3 hours while being stirred. Subsequently, thereaction mixture was concentrated under reduced pressure, and theresulting residue was dissolved in dichloromethane (200 mL). Thedichloromethane solution was washed with a saturated aqueous solution ofsodium chloride (2×50 mL), was dried over anhydrous sodium sulfate, andwas then concentrated under reduced pressure. The resulting residue waspurified on a silica gel column (eluant=ethyl acetate: methanol=10:1) togive 2.04 g of(3R,4S)-[1-benzyloxycarbonyl-4-hydroxypyrrolidin-3-yl]methanol as amilky white syrup-like product.

MS (EI) m/z=251 (M⁺).

Step 2:

(3R,4S)-[1-Benzyloxycarbonyl-4-hydroxypyrrolidin-3-yl]methanol (2.33 g),sodium azide (1.81 g), triphenylphosphine (2.67 g), andN,N-dimethylformamide (46 mL) were mixed with one another. While thismixture was cooled on an ice bath, a dichloromethane solution (10 mL) ofcarbon tetrabromide (3.38 g) was added dropwise. The reaction mixturewas stirred for 13 hours at room temperature and additional 3 hours at60° C., followed by addition of methanol (3 mL) and concentration underreduced pressure. The resulting residue was dissolved in ethyl acetate(200 mL) and was washed with a saturated aqueous solution of sodiumchloride (2×50 mL), followed by drying over anhydrous sodium sulfate andconcentration under reduce pressure. The resulting residue was purifiedon a silica gel column (eluant=ethyl acetate:hexane=2:1) to give 2.18 gof (3R,4S)-3-azidomethyl-1-benzyloxycarbonyl-4-hydroxypyrrolidine as amilky white syrup-like product.

MS (FAB⁺): m/z=277 (MH⁺).

Step 3:

(3R,4S)-3-Azidomethyl-1-benzyloxycarbonyl-4-hydroxypyrrolidine (300 mg)was dissolved in dichloromethane (6 mL). While this solution was cooledon an ice bath, diethylaminosulfur trifluoride (0.43 mL) was addeddropwise. The mixture was stirred at room temperature for 4 hours. Whilethe reaction vessel was cooled on an ice bath, a saturated aqueoussolution of sodium hydrogen carbonate (6 mL) was added and thedichloromethane layer was collected. The collected dichloromethane layerwas washed with a saturated aqueous solution of sodium chloride (2×2mL), was dried over anhydrous sodium sulfate, and was then concentratedunder reduced pressure. The resulting residue was purified on a silicagel column (eluant=hexane:ethyl acetate=2:1) to give 211 mg of a mixtureof (3R,4R)-3-azidomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine and3-azidomethyl-1-benzyloxycarbonyl-3-pyrroline.

Step 4:

Platinum oxide (IV) (50.0 mg) was suspended in ethanol (7 mL) and thesuspension was stirred at room temperature for 30 min in a stream ofhydrogen (provided from a balloon). To this suspension, an ethanolsolution (3 mL) of a mixture (551 mg) of(3R,4R)-3-azidomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine and3-azidomethyl-1-benzyloxycarbonyl-3-pyrroline was added, and the mixturewas stirred at room temperature for 5 hours in a stream of hydrogen(provided from a balloon). Subsequently, the catalyst was removed fromthe reaction mixture by filtration and was washed with ethanol. Thefiltrate and the washing solution were combined and the combined organiclayer was concentrated under reduced pressure. The resulting residue wasthen purified on a silica gel column (eluant=ethyl acetate shifted toethyl acetate: methanol=10:1) to give 313 mg of a mixture of(3S,4R)-3-aminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine and3-aminomethyl-1-benzyloxycarbonyl-3-pyrroline.

Step 5:

A mixture (310 mg) of(3S,4R)-3-aminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine and3-aminomethyl-1-benzyloxycarbonyl-3-pyrroline was dissolved in methanol(4 mL). To this solution, molecular sieves 4A (130 mg) and benzaldehyde(0.13 mL) were sequentially added and the mixture was stirred at roomtemperature for 1 hour. Subsequently, a borane-pyridine complex (0.19mL) was added and the mixture was further stirred at room temperaturefor 4 hours. This was followed by addition of 6 mol/L hydrochloric acid(2 mL) and stirring at room temperature for 1 hour. Subsequently, a 30%aqueous solution of sodium hydroxide was added to make the mixture basicand the mixture was extracted with diethyl ether (3×10 mL). The diethylether extracts were combined and the combined diethyl ether layer wasconcentrated under reduced pressure. The resulting residue was purifiedon a silica gel column (eluant=dichloromethane: methanol=10:1) to give177 mg of(3S,4R)-3-benzylaminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine as apale yellow oil.

MS (FAB⁺): m/z=343 (MH⁺).

HRMS (FAB⁺): Calcd for C₂₀H₂₄FN₂O₂: 343.1822; found: 343.1815.

Step 6:

(3S,4R)-3-Benzylaminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine (170mg) was dissolved in methanol (5 mL). To this solution, molecular sieves3A (160 mg), acetic acid (0.29 mL),[(1-ethoxycyclopropyl)oxy]trimethylsilane (0.40 mL), and sodiumcyanoborohydride (93.5 mg) were added and the mixture was heat-refluxedfor 3 hours while being stirred. Subsequently, insoluble materials wereremoved from the reaction mixture by filtration through a Celite pad.The insoluble materials and the Celite pad were washed with methanol.The filtrate and the washing solution were combined and a 2 mol/Laqueous solution of sodium hydroxide was added to make the combinedorganic layer basic (pH>12). Methanol was then removed under reducedpressure and the residue was extracted with diethyl ether (3×10 mL). Thediethyl ether extracts were combined and the combined diethyl etherlayer was dried over anhydrous sodium sulfate and was then concentratedunder reduced pressure. The resulting residue was purified on a silicagel column (eluant=hexane:ethyl acetate=2:1) to give 166 mg of(3S,4R)-3-(N-benzyl-N-cyclopropyl)aminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidineas a colorless tar-like product.

MS (FAB⁺): m/z=383 (MH⁺).

HRMS (FAB⁺): Calcd for C₂₃H₂₈FN₂O₂: 383.2135; found: 383.2119.

Step 7:

(3S,4R)-3-(N-Benzyl-N-cyclopropyl)aminomethyl-1-benzyloxycarbonyl-4-fluoropyrrolidine(160 mg) was dissolved in ethanol (3 mL). To this solution, 10%palladium carbon (20.0 mg) was added and the mixture was stirred at roomtemperature for hours in a stream of hydrogen (provided from a balloon).Subsequently, the catalyst was collected from the reaction mixture byfiltration through a Celite pad. The collected catalyst and the Celitepad were washed with ethanol. The filtrate and the washing solution werecombined and the resulting residue was purified on a silica gel column(eluant=ethyl acetate: methanol=20:1, shifted to dichloromethane:methanol=10:1) to give 50.7 mg of(3R,4R)-3-cyclopropylaminomethyl-4-fluoropyrrolidine as a colorless oil.

MS (FAB⁺): m/z=159 (MH⁺).

HRMS (FAB⁺): Calcd for C₈H₁₆FN₂: 159.1298; found: 159.1286.

Reference Example 14 Synthesis of(3R,4S)-3-[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl-4-fluoromethylpyrrolidine

Step 1:

(1S,5R)-7-[(1R)-1-Phenylethyl]-3-oxa-7-azabicyclo[3.3.0]octane-2-one(7.73 g, 33.4 mmol) was dissolved in ethanol (92 mL). To this solution,cyclopropylamine (46.3 ml) was added, and the mixture was stirred at 80°C. for 44 hours and was subsequently concentrated under reducedpressure. The resulting residue was dissolved in ethyl acetate (300 mL)and the solution was washed with water (2×50 mL), followed by dryingover anhydrous sodium sulfate and concentration under reduced pressure.To the resulting residue, diisopropyl ether (300 mL) was added and themixture was heated to form crystal and was then concentrated toapproximately 1/2. The formed crystal was collected by filtration andthe collected crystal was washed with diisopropyl ether and was driedunder reduced pressure to give 4.41 g of(3R,4S)-N-cyclopropyl-4-hydroxymethyl-1-[(1S)-1-phenylethyl]pyrrolidine-3-carboxamideas a white crystal. The filtrate and the washing solution were combinedand the combined solvent was concentrated under reduced pressure. Theresulting residue was purified on a silica gel column(eluant=hexane:ethyl acetate=1:1, shifted to ethyl acetate) to obtainadditional 1.50 g of(3R,4S)-N-cyclopropyl-4-hydroxymethyl-1-[(1S)-1-phenylethyl]pyrrolidine-3-carboxamide.The total amount of the compound was 5.91 g.

MS (EI) m/z=288 (M⁺).

Elementary analysis (%): Calcd for C₁₇H₂₄N₂O₂.0.2H₂O: C 69.93, H 8.42, N9.59; found: C, 70.16; H, 8.32; N, 9.60.

Step 2:

(3R,4S)-N-Cyclopropyl-4-hydroxymethyl-1-[(1S)-1-phenylethyl]pyrrolidine-3-carboxamide(7.54 g) was dissolved in N,N-dimethylformamide (180 mL). While thissolution was cooled on an ice bath, imidazole (2.67 g) andtert-butylchlorodimethylsilane (4.72 g) were sequentially added. Themixture was stirred at room temperature for 90 min and was subsequentlyconcentrated under reduced pressure. The resulting residue was dissolvedin ethyl acetate (300 mL) and the solution was washed with water (2×100mL), followed by drying over anhydrous sodium sulfate and concentrationunder reduced pressure. The resulting residue was purified on a silicagel column (eluant=ethyl acetate) to give 7.05 g of(3R,4S)-N-cyclopropyl-4-(tert-butyldimethylsilyl)oxymethyl-1-[(1S)-1-phenylethyl]pyrrolidine-3-carboxamideas a pale yellow tar-like product.

MS (EI) m/z: =402 (M⁺).

Step 3:

(3R,4S)-N-Cyclopropyl-4-(tert-butyldimethylsilyl)oxymethyl-1-[(1S)-1-phenylethyl]pyrrolidine-3-carboxamide(7.00 g) was dissolved in toluene (70 mL). To this solution,borane-dimethyl sulfide complex (2.20 mL) was added and the mixture washeat-refluxed for 5 hours while being stirred. Subsequently, thereaction mixture was allowed to cool to room temperature. Followingaddition of a 10% aqueous solution of sodium carbonate (42 mL), themixture was stirred at 100° C. for 1 hour and the toluene layer wasseparated. The toluene layer was washed with water (2×30 mL), was driedover anhydrous sodium sulfate, and was then concentrated under reducedpressure. The resulting residue was purified on a silica gel column(eluant=hexane:ethyl acetate=4:1) to give 4.78 g of(3S,4S)-4-(tert-butyldimethylsilyl)oxymethyl-3-cyclopropylaminomethyl-1-[(1S)-1-phenylethyl]pyrrolidineas a colorless oil.

Step 4:

(3S,4S)-4-(tert-Butyldimethylsilyl)oxymethyl-3-cyclopropylaminomethyl-1-[(1S)-1-phenylethyl]pyrrolidine(4.70 g) was dissolved in dichloromethane (70 mL). To this solution,di-tert-butyldicarbonate (2.77 g) was added and the mixture was stirredat room temperature for 2 hours. Subsequently, the reaction mixture wasconcentrated under reduced pressure and the resulting residue waspurified on a silica gel column (eluant=hexane:ethyl acetate=4:1,shifted to 1:1) to give 5.28 g of(3R,4S)-3-[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl-4-(tert-butyldimethylsilyl)oxymethyl-1-[(1S)-1-phenylethyl]pyrrolidineas a colorless oil.

Step 5:

Process (A):(3R,4S)-N-Cyclopropyl-4-hydroxymethyl-1-[(1S)-1-phenylethyl]pyrrolidine-3-carboxamide(1.49 g) was dissolved in toluene (15 mL). To this solution, aborane-dimethyl sulfide complex (0.65 mL) was added and the mixture washeat-refluxed for 6 hours while being stirred. Subsequently, thereaction mixture was allowed to cool to room temperature. Followingaddition of a 10% aqueous solution of sodium carbonate (12.4 mL), themixture was stirred at 100° C. for 1 hour and the toluene layer wasseparated. The toluene layer was then washed with water (10 mL) and wasdried over anhydrous sodium sulfate. Following addition ofdi-tert-butyldicarbonate (1.13 g), the mixture was stirred at roomtemperature for 30 min and was subsequently allowed to stand overnight.The reaction mixture was then concentrated under reduced pressure andthe resulting residue was purified on a silica gel column(eluant=hexane:ethyl acetate=1:1) to give 1.50 g of(3R,4S)-3-[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl-4-hydroxymethyl-1-[(1S)-1-phenylethyl]pyrrolidineas pale brown crystal.

Process (B):(3R,4S)-3-[(N-tert-Butoxycarbonyl-N-cyclopropyl)amino]methyl-4-(tert-butyldimethylsilyl)oxymethyl-1-[(1S)-1-phenylethyl]pyrrolidine(3.02 g) was dissolved in tetrahydrofuran (45 mL). While this solutionwas cooled on an ice bath, tetrabutylammonium fluoride (lmol/Ltetrahydrofuran solution, 7.42 ml) was added dropwise and the mixturewas stirred at room temperature for 2 hours. Subsequently, a saturatedaqueous solution of sodium chloride (60 mL) was added and the mixturewas extracted with ethyl acetate (2×150 mL). The ethyl acetate extractswere combined and the combined ethyl acetate layer was washed with asaturated aqueous solution of sodium chloride (2×100 mL), followed bydrying over anhydrous sodium sulfate and concentration under reducedpressure. The resulting residue was dissolved in ethyl acetate (10 mL)and the formed crystal was collected by filtration, was washed with asmall amount of ethyl acetate, and was then dried under reduced pressureto give 781 mg of(3R,4S)-3-[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl-4-hydroxymethyl-1-[(1S)-1-phenylethyl]pyrrolidineas a white crystal. The filtrate and the washing solution were combinedand the combined organic layer was concentrated under reduced pressure.The resulting residue was purified on a silica gel column(eluant=hexane:ethyl acetate=1:1) to give additional 1.43 g of(3R,4S)-3-[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl-4-hydroxymethyl-1-[(1S)-1-phenylethyl]pyrrolidine.The total amount of the compound was 2.21 g.

MS (EI) m/z: =374 (M⁺).

Elementary analysis (%): Calcd for C₂₂H₃₄N₂O₃: C, 70.55; H, 9.15; N,7.48; found: C, 70.56; H, 9.29; N, 7.52.

Step 6:

(3R,4S)-3-[(N-tert-Butoxycarbonyl-N-cyclopropyl)amino]methyl-4-hydroxymethyl-1-[(1S)-1-phenylethyl]pyrrolidine(2.66 g) was dissolved in dichloromethane (40 mL). While this solutionwas cooled on a sodium chloride/ice bath, triethylamine (1.05 mL) wasadded. This was followed by dropwise addition of methanesulfonylchloride (0.58 mL). After being stirred at −5° C. for 30 min, thereaction mixture was washed with water, was dried over anhydrous sodiumsulfate, and was then concentrated under reduced pressure. The resultingresidue was dissolved in tetrahydrofuran (21 mL). To this solution,tetrabutylammonium fluoride (lmol/L tetrahydrofuran solution, 21.3 mL)was added and the mixture was heat-refluxed for 2 hours while beingstirred. The reaction mixture was concentrated under reduced pressureand the resulting residue was dissolved in ethyl acetate (200 mL). Theethyl acetate solution was washed with water (2×50 mL), was dried overanhydrous sodium sulfate, and was then concentrated under reducedpressure. The resulting residue was purified on a silica gel column(eluant=hexane:ethyl acetate=4:1, shifted to 1:1) to give 1.13 g of(3R,4S)-3-[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl-4-fluoromethyl-1-[(1S)-1-phenylethyl]pyrrolidineas a pale brown tar-like product.

MS (EI) m/z=376 (M⁺).

Step 7:

(3R,4S)-3-[(N-tert-Butoxycarbonyl-N-cyclopropyl)amino]methyl-4-fluoromethyl-1-[(1S)-1-phenylethyl]pyrrolidine(1.10 g) was dissolved in methanol (20 mL). To this solution, asuspension of 10% palladium carbon (230 mg) in water (4 mL) and ammoniumformate (921 mg) were sequentially added and the mixture washeat-refluxed for 90 min while being stirred. Subsequently, the catalystwas removed from the reaction mixture by filtration through a Celitepad. The removed catalyst and the Celite pad were washed with ethanolcontaining 20% water. The filtrate and the washing solution werecombined and the combined solution was concentrated under reducedpressure. Water (20 mL) was then added to the resulting residue. Whilethis mixture was cooled on an ice bath, a 30% aqueous solution of sodiumhydroxide was added to make the mixture basic (pH14). The mixture wassubsequently extracted with dichloromethane (50 mL×2). Thedichloromethane extracts were combined washed with water (2×20 mL), andthe combined dichloromethane layer was dried over anhydrous sodiumsulfate and was then concentrated under reduced pressure. The resultingresidue was purified on a silica gel column (eluant=dichloromethane:methanol=20:1) to give 684 mg of(3R,4S)-3-[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl-4-fluoromethylpyrrolidineas a pale brown tar-like product.

MS (EI) m/z=272 (M⁺).

Reference Example 15 Synthesis of(3R,4R)-3-cyclopropylaminomethyl-4-methylpyrrolidine•trifluoroacetate

Step 1:

1-Benzyl-4-(R)-methyl-3-(R)-[(4-(S)-phenyl-2-oxazolidinone-3-yl)carbonyl]pyrrolidine(150 g) was dissolved in cyclopropylamine (650 mL). The mixture wasstirred at room temperature for 23 hours and was subsequentlyconcentrated under reduced pressure. To the resulting residue,diisopropyl ether (800 mL) was added and the mixture was stirred at roomtemperature for 70 min. The resulting crystal was then collected byfiltration. The collected crystal was then dissolved in dichloromethane(800 mL) and the solution was extracted with lmol/L hydrochloric acid(2×400 mL). The 1 mol/L hydrochloric acid extracts were combined. Whilethe combined solution was cooled on an ice bath, a 30% aqueous solutionof sodium hydroxide was added to make the solution basic (pH13). Theresulting crystal was collected by filtration, was sequentially washedwith water and diisopropyl ether, and was then dried under reducedpressure to give 52.2 g of(3R,4R)-1-1-benzyl-N-cyclopropyl-4-methyl-3-pyrrolidinecarboxamide as awhite crystal.

Step 2:

(3R,4R)-1-Benzyl-N-cyclopropyl-4-methyl-3-pyrrolidinecarboxamide (70.0g) was dissolved in toluene (700 mL). While this solution was cooled onan ice bath, a borane-dimethyl sulfide complex (90%, 34.3 mL) was addeddropwise. The mixture was then stirred for 15 min and was heat-refluxed.After the reaction mixture was cooled to room temperature, a 10% aqueoussolution of Na₂CO₃ (400 mL) was added and the mixture was stirred at100° C. for 2 hours. The mixture was cooled to room temperature and thetoluene layer was separated. The toluene layer was then washed withwater (2×250 mL), was dried over anhydrous sodium sulfate, and wasconcentrated under reduced pressure. The resulting residue was purifiedby distillation under reduced pressure to obtain(3S,4R)-1-benzyl-3-cyclopropylaminomethyl-4-methylpyrrolidine (62.1 g)as a colorless oil.

Step 3:

(3S,4R)-1-Benzyl-3-cyclopropylaminomethyl-4-methylpyrrolidine (25.0 g)was dissolved in ethanol (200 mL). To this solution, trifluoroaceticacid (15.7 mL) and 10% palladium carbon (12.5 g) were added and themixture was stirred at room temperature for 9 hours under a hydrogenpressure of 3.9×10⁵ Pa. The catalyst was collected from the reactionmixture by filtration and was washed with ethanol containing 25% water(300 mL). The filtrate and the washing solution were combined and thecombined solution was concentrated under reduced pressure. The resultantpale brown crystal was suspended in tetrahydrofuran (100 mL) and wascollected by filtration. The collected crystal was washed withtetrahydrofuran and was dried under reduced pressure to give 34.1 g of(3R,4R)-3-cyclopropylaminomethyl-4-methylpyrrolidine-trifluoroacetate asa white crystal.

Example 1 Synthesis of(3R)-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

Process (A):[(3R)-9,10-Difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]difluoroboron (51.0 g), 3(R)-cyclopropylaminomethylpyrrolidine (24.7g), triethylamine (24.6 mL) and dimethylsulfoxide (500 mL) were mixedwith one another and the mixture was stirred at 70° C. for 1 hour.Subsequently, the reaction mixture was concentrated under reducedpressure and the resulting residue was purified on a silica gel column(eluant=dichloromethane: methanol=10:1). The eluates were combined andthe combined solution was concentrated under reduced pressure. To theresulting residue, 80% ethanol (2500 mL) and triethylamine (25.0 mL)were added and the mixture was heat-refluxed for 2 hours while beingstirred. Subsequently, the reaction mixture was left on an ice bath for2 hours and the resulting crystal was collected by filtration. Thecollected crystal was washed with ethanol, was suspended in purifiedwater (300 ml), and was then collected by filtration. The collectedcrystal was dried under reduced pressure and was purified on a silicagel column (eluant=dichloromethane: methanol=10:1). The eluates werecombined and the combined solution was concentrated under reducedpressure. The resulting residue was dissolved in ethanol (2000 mL) byheating and the solution was allowed to stand for 14 hours at roomtemperature. The resultant crystal was collected by filtration and thecollected crystal was washed with ethanol and was dried under reducedpressure to give 27.7 g of (3 R)-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow powder.

Process B: To a dichloromethane solution (273 mL) ofbis(acetato-O)[(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (22.6 g), (3R)-3-cyclopropylaminomethylpyrrolidine (8.41 g) andtriethylamine (7.59 g) were added and the mixture was allowed to standat room temperature for 13 hours. Subsequently, the reaction mixture wassequentially washed with water (200 mL) and a saturated aqueous solutionof sodium chloride (50 ml), was dried over anhydrous sodium sulfate, andwas then concentrated under reduced pressure. The resulting residue waspurified on a silica gel column (dichloromethane: methanol=15:1) toobtain a yellow amorphous product. To this product, a 5% aqueoussolution of acetic acid (100 mL) was added and the mixture was stirredat 80° C. for 3 hours. Subsequently, the reaction mixture was washedwith ethyl acetate (100 mL). While the mixture was cooled on an icebath, a 1 mol/L aqueous solution of sodium hydroxide was added to adjustthe pH to 7.01 and the mixture was further stirred for 0.5 hours. Theresultant crystal was collected by filtration, was washed with purifiedwater (2×50 mL), and was then dissolved in ethanol (1200 mL) by heating.The solution was allowed to stand at room temperature for 12 hours.Subsequently, the resulting crystal was collected by filtration,followed by washing with ethanol and drying under reduced pressure, togive 11.2 g of(3R)-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow crystal.

MS (EI) m/z: 419 (M⁺).

Elementary analysis (%): Calcd for C₂₁H₂₃F₂N₃O₄: C, 60.14; H, 5.53; N,10.02; found: C, 60.01; H, 5.47; N, 9.94.

Example 2 Synthesis of(3R)-10-[(3R)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1, bis(acetato-O)[(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷ boron (982 mg) was reacted with(3S)-3-cyclopropylaminomethylpyrrolidine (335 mg) to give 587 mg of(3R)-10-[(3R)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow crystal.

MS (FAB⁺) m/z: 420 (MH⁺).

Elementary analysis (%): Calcd for C₂₁H₂₃F₂N₃O₄.0.25H₂O: C, 59.50; H,5.59; N, 9.91; found: C, 59.68; H, 5.47; N, 9.97.

Example 3 Synthesis of(3R)-10-[3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1,bis(acetato-O)[(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (513 mg) was reacted with 3-cyclopropylaminomethylpyrrolidine(185 mg) to give 231 mg of(3R)-10-(3-cyclopropylaminomethyl-1-pyrrolidinyl)-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow crystal.

MS (FAB⁺) m/z: 420 (MH⁺).

Elementary analysis (%): Calcd for C₂₁H₂₃F₂N₃O₄.0.25H₂O: C, 59.50, H,5.59, N, 9.91: found: C, 59.41; H, 5.41; N, 9.89.

Example 4 Synthesis of(3S)-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methoxymethyl-1-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1, bis(acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methoxymethyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (790 mg) was reacted with(3R)-3-cyclopropylaminomethylpyrrolidine (303 mg) to give 602 mg of(3S)-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methoxymethyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow crystal.

MS (FAB⁺) m/z: 432 (MH⁺).

Elementary analysis (%): Calcd for C₂₂H₂₆FN₃O₅: C, 61.24; H, 6.07; N,9.74; found: C, 61.01; H, 6.04; N, 9.73.

Example 5 Synthesis of(3S)-3-acetoxymethyl-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1, bis(acetato-O)[(3S)-3-acetoxymethyl-9,10-difluoro-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (934 mg) was reacted with(3R)-3-cyclopropylaminomethylpyrrolidine (337 mg) to give 612 mg of(3S)-3-acetoxymethyl-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow crystal.

MS (FAB⁺) m/z: 460 (MH⁺).

Elementary analysis (%): Calcd for C₂₃H₂₆FN₃O₆.H₂O: C 57.85, H 5.91, N8.80; found: C, 57.94; H, 5.83; N, 8.89.

Example 6 Synthesis of(3S)-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-hydroxymethyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

A 1 mol/L aqueous solution of sodium hydroxide (8.0 mL) containing(3S)-3-acetoxymethyl10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid (368 mg) was stirred at 50° C. for 2 hours. While this mixture wascooled on an ice bath, lmol/L hydrochloric acid was added to adjust thepH to 7.05 and the mixture was further stirred for 0.5 hours. Theresultant crystal was collected by filtration, was washed with purifiedwater, and was then dissolved in ethanol (50 mL) by heating. Thesolution was allowed to stand at room temperature for 2 hours.Subsequently, the resulting crystal was collected by filtration, waswashed ethanol, and was then dried under reduced pressure to give 251 mgof(3S)-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-hydroxymethyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow crystal.

MS (FAB⁺) m/z: 418 (MH⁺).

Elementary analysis (%): Calcd for C₂₁H₂₄FN₃O₅.0.5H₂O: C 59.15, H 5.91,N 9.85; found: C, 59.16; H, 5.92; N, 9.88.

Example 7 Synthesis of10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methylene-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

(3R)-10-[(3S)-3-Cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid (252 mg) was suspended in ethanol (1 mL). To this suspension, a 1mol/L aqueous solution of sodium hydroxide (6 mL) was added and themixture was stirred at room temperature for 2 hours. Subsequently, thereaction mixture was concentrated under reduced pressure and theresulting residue was dissolved in purified water (10 ml). While thissolution was cooled on an ice bath, 1 mol/L hydrochloric acid was addedto adjust the pH to 7.03 and the mixture was further stirred for 0.5hours. The resultant crystal was collected by filtration to obtain 214mg of10-[(3S)-cyclopropylmethylamino-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methylene-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow powder.

MS (FAB⁺) m/z: 400 (MH⁺).

Elementary analysis (%): Calcd for C₂₂H₂₃FN₃O₄.1.75H₂O: C, 58.53; H,5.96; N, 9.75; found: C, 58.62; H, 5.79; N, 9.76.

Example 8 Synthesis of(3R)-10-[(3S)-cyclopropylaminomethyl-1-pyrrolidinyl]-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1, bis(acetato-O)[(3R)-10-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (500 mg) was reacted with(3R)-3-cyclopropylaminomethylpyrrolidine (240 mg) to give 335 mg of(3R)-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow needle-shaped product.

MS (FAB⁺) m/z: 402 (MH⁺).

Elementary analysis (%): Calcd for C₂₁H₂₄FN₃O₄: C, 62.83; H, 6.03; N,10.47; found: C, 62.56; H, 5.94; N, 10.40.

Example 9 Synthesis of(3S)-10-[(3S)-cyclopropylaminomethyl-1-pyrrolidinyl]-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1, bis(acetato-O)[(3S)-10-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (1000 mg) was reacted with(3R)-3-cyclopropylaminomethylpyrrolidine (431 mg) to give 335 mg of(3S)-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow needle-shaped product.

MS (EI⁺) m/z: 383 (M⁺).

Elementary analysis (%): Calcd for C₂₁H₂₅N₃O₄: C, 65.78; H, 6.57; N,10.96; found: C, 65.58, H 6.61, N 10.91.

Example 10 Synthesis of(3S)-10-(trans-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl)-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1, bis(acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (300 mg) was reacted withtrans-3-cyclopropylaminomethyl-4-methylpyrrolidine (136 mg) to give 166mg of(3S)-10-(trans-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl)-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as yellow crystal.

MS (EI⁺) m/z: 415 (M⁺).

Elementary analysis (%): Calcd for C₂₂H₂₆FN₃O₄.0.5H₂O: C 62.25, H 6.41,N 9.90; found: C, 62.30; H, 6.17; N, 10.06.

Example 11 Synthesis of(3S)-10-[(3S,4R)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1, bis(acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (500 mg) was reacted with(3R,4R)-3-cyclopropylamino-4-methylpyrrolidine (226 mg) to give 362 mgof(3S)-10-[(3S,4R)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow crystal.

MS (EI⁺) m/z: 415 (M⁺)

Elementary analysis (%): Calcd for C₂₂H₂₆FN₃O₄: C, 63.60; H, 6.31; N,10.11; found: C, 63.41; H, 6.30; N, 10.17.

Example 12 Synthesis of(3S)-10-[(3R,4S)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1,bis(acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (500 mg) was reacted with(3S,4S)-3-cyclopropylamino-4-methylpyrrolidine (226 mg) to give 276 mgof(3S)-10-[(3R,4S)-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a pale yellow crystal.

MS (EI⁺) m/z: 415 (M⁺).

Elementary analysis (%): Calcd for C₂₂H₂₆FN₃O₄.0.5H₂O: C, 62.25; H,6.41; N, 9.90; found: C, 62.23; H, 6.06; N, 9.92.

Example 13 Synthesis of(3S)-10-(cis-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl)-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1,bis(acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (220 mg) was reacted withcis-3-cyclopropylaminomethyl-4-methylpyrrolidine (100 mg) to give 109 mgof(3S)-10-(cis-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl)-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow crystal.

MS (EI⁺) m/z: 415 (M⁺).

Elementary analysis (%): Calcd for C₂₂H₂₆FN₃O₄.H₂O: C 60.96, H 6.51, N9.69; found: C, 61.27; H, 6.69; N, 9.52.

Example 14 Synthesis of(3S)-10-[(3S,4S)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1, bis(acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (1000 mg) was reacted with(3R,4S)-cyclopropylamino-4-methylpyrrolidine (452 mg) to give 474 mg of(3S)-10-[(3S,4S)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow crystal.

MS (EI⁺) m/z: 415 (M⁺).

Elementary analysis (%): Calcd for C₂₂H₂₆FN₃O₄.0.25H₂O: C, 62.92; H,6.36; N, 10.01; found: C, 62.69; H, 6.52; N, 9.98.

Example 15 Synthesis of(3S)-10-[(3R,4R)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1,bis(acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (250 mg) was reacted with(3S,4R)-3-cyclopropylamino-4-methylpyrrolidine (113 mg) to give 33 mg of(3S)-10-[(3R,4R)-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a pale yellow crystal.

MS (EI⁺) m/z: 415 (M⁺).

Elementary analysis (%): Calcd for C₂₂H₂₆FN₃O₄.0.5H₂O: C 62.25, H 6.41,N 9.90; found: C, 61.98; H, 6.57; N, 9.91.

Example 16 Synthesis of(3R)-10-(trans-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl)-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1,bis(acetato-O)[(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (300 mg) was reacted withtrans-3-cyclopropylaminomethyl-4-methylpyrrolidine (130 mg) to give 110mg of(3R)-10-(trans-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl)-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid.

MS (EI⁺) m/z: 433 (M⁺).

Elementary analysis (%): Calcd for C₂₂H₂₅F₂N₃O₄: C, 60.96; H, 5.81; N,9.69; found: C, 60.81; H, 5.85; N, 9.66.

Example 17 Synthesis of(3R)-10-[(3S,4R)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1,bis(acetato-O)[(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (1000 mg) was reacted with(3R,4R)-cyclopropylamino-4-methylpyrrolidine (397 mg) to give 620 mg of(3R)-10-[(3S,4R)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow crystal.

MS (EI⁺) m/z: 433 (M⁺).

Elementary analysis (%): Calcd for C₂₂H₂₅F₂N₃O₄: C, 60.96; H, 5.81; N,9.69; found: C, 60.81; H, 5.86; N, 9.63.

Example 18 Synthesis of(3R)-10-[(3S,4S)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1, bis(acetato-O)[(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (500 mg) was reacted with(3R,4R)-cyclopropylamino-4-methylpyrrolidine (199 mg) to give 422 mg of(3R)-10-[(3S,4S)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid as a yellow crystal.

MS (EI⁺) m/z: 433 (M⁺).

Elementary analysis (%): Calcd for C₂₂H₂₅F₂N₃O₄: C, 60.96; H, 5.81; N,9.69; found: C, 60.79; H, 5.91; N, 9.77.

Example 19 Synthesis of(3S)-10-(trans-3-cyclopropylaminomethyl-4-trifluoromethyl-1-pyrrolidinyl)-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1, bis(acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (300 mg) was reacted withtrans-3-cyclopropylaminomethyl-4-trifluoromethylpyrrolidine (198 mg) togive 87 mg of(3S)-10-(trans-3-cyclopropylaminomethyl-4-trifluoromethyl-1-pyrrolidinyl)-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid.

MS (FAB⁺) m/z: 470 (MH⁺).

Elementary analysis (%): Calcd for C₂₂H₂₃F₄N₃O₄: C 56.29, H 4.94, N8.95; found: C, 55.97; H, 4.84; N, 9.00.

Example 20 Synthesis of(3R)-10-[(3S,4S)-3-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1, bis(acetato-O)[(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (500 mg) was reacted with(3R,4S)-3-cyclopropylaminomethyl-4-fluoropyrrolidine (204 mg) to give387 mg of(3R)-10-[(3S,4S)-3-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid as a pale yellow crystal.

MS (FAB⁺):m/z=438 (MH⁺).

Elementary analysis (%): Calcd for C₂₁H₂₂F₃N₃O₄: C, 57.66; H, 5.07; N,9.61; found: C, 57.47; H, 5.07; N, 9.57.

Example 21 Synthesis of(3S)-10-[(3S,4S)-3-cyclopropylamino-4-fluoro-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1,bis(acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (64.6 mg) was reacted with(3R,4S)-3-cyclopropylaminomethyl-4-fluoropyrrolidine (25.0 mg) to give18.5 mg of(3R)-10-[(3S,4S)-3-cyclopropylamino-4-fluoro-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid as a yellow powder.

MS (FAB⁺) m/z=420 (MH⁺).

HRMS (FAB⁺): Calcd for C₂₁H₂₄F₂N₃O₄: 420.1735; found: 420.1747.

Example 22 Synthesis of(3R)-10-[(3S,4S)-3-[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl-4-fluoromethyl-1-pyrrolidine]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid

Bis(acetato-O)[(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (912 mg),(3R,4S)-3-[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl-4-fluoromethylpyrrolidine(640 mg), triethylamine (0.33 mL) and acetonitrile (17 mL) were mixedwith one another and the mixture was stirred at 60° C. for 90 min.Subsequently, the reaction mixture was concentrated under reducedpressure and the resulting residue was purified on a silica gel column(eluant=ethyl acetate: methanol=20:1). To the eluate, a 5% aqueoussolution of acetic acid (17 mL) and ethanol (10 mL) were added and themixture was stirred at 80° C. for 2 hours. The mixture was allowed tocool and the resulting crystal was collected by filtration, was washedwith a mixture of water and ethanol, and was then dried under reducedpressure to give 915 mg of(3R)-10-[(3S,4S)-3-[(N-tert-butoxycarbonyl-N-cyclopropyl)amino]methyl-4-fluoromethyl-1-pyrrolidine]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylic acid as a yellow powder.

MS (EI) m/z=551 (M⁺).

Elementary analysis (%): Calcd for C₂₇H₃₂F₃N₃O₆.0.5H₂O: C, 57.85; H,5.93; N, 7.50; found: C, 57.90; H, 5.80; N, 7.49.

Example 23 Synthesis of(3R)-10-[(3S,4S)-3-cyclopropyl]amino]methyl-4-fluoromethyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid hydrochloride

(3R)-10-[(3S,4S)-3-[(N-tert-Butoxycarbonyl-N-cyclopropyl)amino]methyl-4-fluoromethyl-1-pyrrolidine]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid (860 mg) was dissolved in ethanol (9 mL) saturated with hydrogenchloride. The mixture was stirred at room temperature for 1 hour and wassubsequently concentrated under reduced pressure. To the resultingresidue, ethanol (50 mL) was added and the mixture was concentratedunder reduced pressure. After repeating the ethanol addition andconcentration once, ethanol (50 mL) was added to the resultant residue,and the mixture was heated to 70° C. and was then allowed to stand atroom temperature for 1 hour. The resulting crystal was collected byfiltration, followed by washing with ethanol and drying under reducedpressure, to give 762 mg of(3R)-10-[(3S,4S)-3-cyclopropyl]amino]methyl-4-fluoromethyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid hydrochloride as a yellow crystal.

MS (FAB⁺): m/z=452 (MH⁺).

Elementary analysis (%): Calcd for C₂₂H₂₄F₃N₃O₄.HCl.H₂O.0.5C₂H₅OH: C,52.23; H, 5.72; N, 7.94; found: C, 52.17; H, 5.38; N, 8.20.

Example 24 Synthesis of(3R)-10-[(3S,4R)-3-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid

In a similar manner to Process (B) in Example 1,bis(acetato-O)[(3R)-9,10-difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylato-O⁶,O⁷]boron (100 mg) was reacted with(3R,4R)-3-cyclopropylaminomethyl-4-fluoropyrrolidine (40.6 mg) to give71.0 mg of(3R)-10-[(3S,4R)-3-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid as a pale yellow crystal.

MS (FAB⁺): m/z=438 (MH⁺).

Elementary analysis (%): Calcd for C₂₁H₂₂F₃N₃O₄: C, 57.66; H, 5.07; N,9.61; found: C, 57.50; H, 5.18; N, 9.22.

Example 25 Synthesis of(3R)-10-[(3S,4S)-3-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid methanesulfonate

(3R)-10-[(3S,4S)-3-Cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid (50.0 mg) was suspended in ethanol (2 mL). To this suspension,methanesulfonic acid (15.0 μL) was added and the mixture was stirred atroom temperature for 1 hour. The resulting crystal was collected byfiltration, was washed with ethanol, and was then dried under reducedpressure to give 50.4 mg of(3R)-10-[(3S,4S)-3-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid methanesulfonate as a pale yellow crystal.

MS (FAB⁺): m/z=438 (MH⁺).

Elementary analysis (%): Calcd for C₂₁H₂₂F₃N₃O₄.CH₃SO₃H.0.25H₂O: C49.11, H 4.96, N 7.81; found: C, 49.18; H, 4.86; N, 7.42.

Example 26 Synthesis of(3R)-10-[(3S,4S)-3-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid hydrochloride

(3R)-10-[(3S,4S)-3-Cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid (50.0 mg) was suspended in ethanol (2 mL). To this suspension,ethanol (60.0 μL) saturated with hydrogen chloride was added and themixture was stirred at room temperature for 1 hour. The resultingcrystal was collected by filtration, was washed with ethanol, and wasthen dried under reduced pressure to give 52.9 mg of(3R)-10-[(3S,4S)-3-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d.e][1,4]benzoxazine-6-carboxylicacid hydrochloride as a pale yellow crystal.

MS (FAB⁺): m/z=438 (MH⁺).

Elementary analysis (%): Calcd for C₂₁H₂₂F₃N₃O₄.HCl.0.25H₂O: C 52.73, H4.95, N 8.78; found: C, 52.68; H, 5.04; N, 8.28.

(Antibacterial Activity)

Test Example 1 In Vitro Antibacterial Activity

The in vitro antibacterial activity (as measured by the minimuminhibitory concentration (MIC)) was determined for each of the compoundsof the present invention by the agar dilution method according to NCCLS(National Committee for Clinical Laboratory Standard (1997), Methods forDilution Antibacterial Susceptibility Tests for Bacteria that growAerobically-Forth Edition: Approved Standard m7-A4. NCCLS, Villanova,Pa.), which involved the use of Muller-Hinton agar medium. Forpneumococci and enterococci, MIC was determined by using Muller-Hintonagar medium containing 5% defibrinated equine blood. The results areshown in Table 1 below. TABLE 1 in vitro antibacterial activity MIC(mg/mL) Strain Example 1 Example 2 Example 4 Example 5 Example 6 S.aureus Smith 0.016 0.031 0.031 0.25 0.25 S. aureus MR5867 0.016 0.0160.031 0.25 0.25 S. aureus MS16401 0.125 0.25 0.5 4 4 S. pneumoniae TypeIII 0.032 0.125 0.125 0.125 0.125 E. faecalis IID682 0.063 0.25 0.250.25 0.25 MIC (mg/mL) Example Example Example Example Strain Example 710 11 12 13 S. aureus Smith ≦0.008 0.016 0.008 0.016 0.008 S. aureusMR5867 ≦0.008 0.016 0.008 0.016 0.008 S. aureus MS16401 0.063 0.0630.031 0.063 0.031 S. pneumoniae Type III 0.016 0.031 0.016 0.063 0.031E. faecalis IID682 0.125 0.125 0.063 0.25 0.125 MIC (mg/mL) ExampleExample Example Example Example Strain 14 15 16 17 18 S. aureus Smith0.008 0.031 0.016 0.008 0.008 S. aureus MR5867 0.008 0.031 0.016 0.0080.016 S. aureus MS16401 0.031 0.063 0.063 0.063 0.063 S. pneumoniae TypeIII ≦0.008 0.063 0.032 0.016 0.016 E. faecalis IID682 0.063 0.125 0.0630.063 0.063 MIC (mg/mL) Example Example Example Strain 19 20 21Ciprofloxacin S. aureus Smith 0.016 0.008 0.016 0.25 S. aureus MR58670.016 0.016 0.016 0.25 S. aureus MS16401 0.125 0.063 0.031 8 S.pneumoniae Type III 0.125 0.016 0.016 0.5 E. faecalis IID682 0.25 0.0630.063 0.5S. aureus MR5867: methicillin-resistant S. aureusS. aureus MS16401: quinolone-resistant S. aureus

INDUSTRIAL APPLICABILITY

The novel 10-(3-cyclopropylaminomethyl-1-pyrrolidinyl)pyridobenzoxazinecarboxylic acid derivatives, salts and hydrates thereof, which arecompounds of the present invention, are not only safe and exhibit strongantibacterial activities, but they are also effective againstdrug-resistant bacteria that are less susceptible to conventionalantibacterial agents.

1. A pyridobenzoxazine carboxylic acid derivative as represented by thefollowing general formula (I), or a salt or a hydrate thereof:

wherein R1 is a methyl group, a fluoromethyl group, a methoxymethylgroup, an acetoxymethyl group, a hydroxymethyl group or a methylene; R2is a hydrogen atom, a lower alkyl group having 1 to 3 carbon atoms, or apharmaceutically acceptable cation and an ester of a prodrug; R3 is ahydrogen atom or a halogen atom; R4 is a hydrogen atom, a lower alkylgroup having 1 to 3 carbon atoms, a fluoromethyl group, atrifluoromethyl group or a fluorine atom; and R5 is a hydrogen atom or afluorine atom.
 2. The compound according to claim 1, a salt or a hydratethereof, wherein R1 in the general formula (I) is a fluoromethyl group.3. The compound according to claim 1, a salt or a hydrate thereof,wherein in the general formula (I), R1 is a fluoromethyl group and R3 isa fluorine atom.
 4. The compound according to claim 1, a salt or ahydrate thereof, wherein in the general formula (I), R1 is afluoromethyl group, R3 is a fluorine atom, and R4 is a hydrogen atom, amethyl group, a fluoromethyl group or a fluorine atom.
 5. The compoundaccording to claim 1, a salt or a hydrate thereof, wherein in thegeneral formula (I), R1 is a fluoromethyl group, R3 is a fluorine atom,R4 is a hydrogen atom, a methyl group, a fluoromethyl group or afluorine atom, and R5 is a hydrogen atom or a fluorine atom.
 6. Thecompound according to claim 1, a salt or a hydrate thereof, wherein R1in the general formula (I) is a methyl group.
 7. The compound accordingto claim 1, a salt or a hydrate thereof, wherein in the general formula(I), R1 is a methyl group, and R3 is a fluorine atom.
 8. The compoundaccording to claim 1, a salt or a hydrate thereof, wherein in thegeneral formula (I), R1 is a methyl group, R3 is a fluorine atom, and R4is a hydrogen atom, a methyl group, a fluoromethyl group or a fluorineatom.
 9. The compound according to claim 1, a salt or a hydrate thereof,wherein in the general formula (I), R1 is a methyl group, R3 is afluorine atom, R4 is a hydrogen atom, a methyl group, a fluoromethylgroup or a fluorine atom, and R5 is a hydrogen atom or a fluorine atom.10. The compound according to claim 1, a salt or a hydrate thereof,wherein the compound of the general formula (I) has a singlestereochemisry.
 11. The compound according to claim 1, a salt or ahydrate thereof, wherein the compound of the general formula (I) is(3S)-10-[(3S,4R)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-3-methyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid, a salt or a hydrate thereof.
 12. The compound according to claim1, a salt or a hydrate thereof, wherein the compound of the generalformula (I) is(3S)-10-[(3S,4S)-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-3-methyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid, a salt or a hydrate thereof.
 13. The compound according to claim1, a salt or a hydrate thereof, wherein the compound of the generalformula (I) is(3R)-10-[(3S,4R)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid, a salt or a hydrate thereof.
 14. The compound according to claim1, a salt or a hydrate thereof, wherein the compound of the generalformula (I) is(3R)-10-[(3S,4S)-3-cyclopropylaminomethyl-4-methyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid, a salt or a hydrate thereof.
 15. The compound according to claim1, a salt or a hydrate thereof, wherein the compound of the generalformula (I) is(3R)-10-[(3S)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid, a salt or a hydrate thereof.
 16. The compound according to claim1, a salt or a hydrate thereof, wherein the compound of the generalformula (I) is(3R)-10-[(3R)-3-cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid, a salt or a hydrate thereof.
 17. The compound according to claim1, a salt or a hydrate thereof, wherein the compound of the generalformula (I) is(3S)-10-[(3S,4R)-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-methyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid, a salt or a hydrate thereof.
 18. The compound according to claim1, a salt or a hydrate thereof, wherein the compound of the generalformula (I) is(3S)-10-[(3S,4S)-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-methyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid, a salt or a hydrate thereof.
 19. The compound according to claim1, a salt or a hydrate thereof, wherein the compound of the generalformula (I) is(3R)-10-[(3S,4R)-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid, a salt or a hydrate thereof.
 20. The compound according to claim1, a salt or a hydrate thereof, wherein the compound of the generalformula (I) is(3R)-10-[(3S,4S)-cyclopropylaminomethyl-4-fluoro-1-pyrrolidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylicacid, a salt or a hydrate thereof.
 21. An antibacterial agent containingas an active ingredient the compound according to claim 1, a salt or ahydrate thereof.